Methods and compositions for treatment of hunter syndrome

ABSTRACT

The present invention provides, among other things, improved methods for purifying I2S protein produced recombinantly for enzyme replacement therapy. The present invention is, in part, based on the surprising discovery that recombinant I2S protein can be purified from unprocessed biological materials, such as, I2S-containing cell culture medium, using a process involving as few as four chromatography columns.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/829,706, filed on Mar. 14, 2013, which claims benefit of U.S.Provisional Patent Application No. 61/666,733, filed Jun. 29, 2012, eachof which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listingsubmitted in electronic form as an ASCII.txt file named“2006685-0278_SEQ_LIST” on Mar. 14, 2013. The .txt file was generated onMar. 5, 2013 and is 15 KB in size. The entire contents of the SequenceListing are herein incorporated by reference.

BACKGROUND

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is anX-chromosome-linked recessive lysosomal storage disorder that resultsfrom a deficiency in the enzyme iduronate-2-sulfatase (I2S). I2S cleavesthe terminal 2-O-sulfate moieties from the glycosaminoglycans (GAG)dermatan sulfate and heparan sulfate. Due to the missing or defectiveI2S enzyme in patients with Hunter syndrome, GAG progressivelyaccumulate in the lysosomes of a variety of cell types, leading tocellular engorgement, organomegaly, tissue destruction, and organ systemdysfunction.

Generally, physical manifestations for people with Hunter syndromeinclude both somatic and neuronal symptoms. For example, in some casesof Hunter syndrome, central nervous system involvement leads todevelopmental delays and nervous system problems. While the non-neuronalsymptoms of Hunter Syndrome are generally absent at birth, over time theprogressive accumulation of GAG in the cells of the body can have adramatic impact on the peripheral tissues of the body. GAG accumulationin the peripheral tissue leads to a distinctive coarseness in the facialfeatures of a patient and is responsible for the prominent forehead,flattened bridge and enlarged tongue, the defining hallmarks of a Hunterpatient. Similarly, the accumulation of GAG can adversely affect theorgan systems of the body. Manifesting initially as a thickening of thewall of the heart, lungs and airways, and abnormal enlargement of theliver, spleen and kidneys, these profound changes can ultimately lead towidespread catastrophic organ failure. As a result, Hunter syndrome isalways severe, progressive, and life-limiting.

Enzyme replacement therapy (ERT) is an approved therapy for treatingHunter syndrome (MPS II), which involves administering exogenousreplacement I2S enzyme to patients with Hunter syndrome.

SUMMARY OF THE INVENTION

The present invention provides, among other things, improved methods forpurifying I2S protein produced recombinantly for enzyme replacementtherapy. The present invention is, in part, based on the surprisingdiscovery that recombinant I2S protein can be purified from unprocessedbiological materials, such as, I2S-containing cell culture medium, usinga process involving as few as four chromatography columns. Approvedexisting purification process of recombinant I2S for enzyme replacementtherapy involves 6 chromatography columns. As described in the Examplessection, recombinant I2S proteins purified using a four-column processaccording to the invention conforms with the marketing purityrequirements in the US and many other countries. In addition, therecombinant I2S enzyme purified according to the present inventionretains high percentage of C_(α)-formylglycine (FGly) (e.g., higher than70% and up to 100%), which is important for the activity of I2S enzyme,and distinct characteristics such as sialic acid content and glycan mapthat may facilitate bioavailability and/or lysosomal targeting of therecombinant I2S protein. Therefore, the present invention provides aneffective, cheaper, and faster process for purifying recombinant I2Sprotein. The present invention is particularly useful for purifyingrecombinant I2S protein produced in serum-free medium.

Thus, in one aspect, the present invention provides a method ofpurifying recombinant I2S protein from an impure preparation using aprocess based on one or more of anion-exchange chromatography,cation-exchange chromatography, mixed-mode chromatography, andhydrophobic interaction chromatography. In some embodiments, aninventive method according to the present invention involves less than 6(e.g., less than 5, less than 4, or less than 3) chromatography steps.In some embodiments, an inventive method according to the presentinvention involves 2, 3, 4 or 5 chromatography steps. In someembodiments, an inventive method according to the present inventioninvolves 4 chromatography steps. In some embodiments, the purifiedrecombinant I2S protein according to the present invention contains lessthan 100 ng/mg Host Cell Protein (HCP) (e.g., less than 90 ng/mg HCP,less than 80 ng/mg HCP, less than 70 ng/mg HCP, less than 60 ng/mg HCP,less than 50 ng/mg HCP, less than 40 ng/mg HCP, less than 30 ng/mg HCP,less than 20 ng/mg HCP, less than 10 ng/mg HCP).

In some embodiments, a suitable anion-exchange chromatography is Qchromatography. In some embodiments, a suitable cation-exchangechromatography is SP chromatography. In some embodiments, a suitablemixed-mode chromatography is hydroxyapatite (HA) chromatography. In someembodiments, a suitable hydrophobic interaction chromatography is phenylchromatography.

It is contemplated that anion-exchange chromatography (e.g., O column),cation-exchange chromatography (e.g., SP column), mixed-modechromatography (e.g., HA column), and hydrophobic interactionchromatography (e.g., phenyl column) can be carried out in any order. Insome embodiments, a method according to the present invention carriesout anion-exchange chromatography (e.g., O column), cation-exchangechromatography (e.g., SP column), mixed-mode chromatography (e.g., HAcolumn), and hydrophobic interaction chromatography (e.g., phenylcolumn) in that order.

In some embodiments, an impure preparation or an intermediate eluate orflow-through is adjusted to pH of about 5.0-7.0 (e.g., about 5.0, 5.5,6.0, 6.5 or 7.0) and the conductivity of about 10-20 mS/cm (e.g., 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mS/cm) prior to loading to theanion-exchange chromatography column (e.g., Q column). In someembodiments, the pH is adjusted using 1M sodium acetate. In someembodiments, the conductivity is adjusted using 5 M sodium chloride. Insome embodiments, the anion-exchange chromatography column, once loaded,is washed using a wash buffer comprising salt (e.g., NaCl) concentrationranging from about 140 mM to 200 mM (e.g., about 140 mM, 145 mM, 150 mM,155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM,or 200 mM) with pH of about 5.0-7.0 (e.g., about 5.0, 5.5, 6.0, 6.5 or7.0). In some embodiments, the anion-exchange chromatography column iseluted using a elution buffer comprising a linear salt (e.g., NaCl)gradient. In some embodiments, a suitable linear NaCl gradient containsa range from about 0-500 mM NaCl (e.g., about 0-400 mM, about 0-350 mM,about 0-300 mM, about 50-500 mM, about 150-500 mM, about 150-450 mM,about 150-400 mM).

In some embodiments, an impure preparation or an intermediate eluate orflow-through is adjusted to conductivity ranging between about 1 mS/cmand 20 mS/cm (e.g., between about 1 mS/cm and 15 mS/cm, between about 1mS/cm and 10 mS/cm, between about 1 mS/cm and 8 mS/cm, between about 1mS/cm and 6 mS/cm, between about 1 mS/cm and 4 mS/cm, between about 2mS/cm and 4 mS/cm) prior to loading to the cation-exchangechromatography column (e.g., SP column). In some embodiments, an impurepreparation or an intermediate eluate or flow-through is adjusted toconductivity ranging between about 2 mS/cm and 4 mS/cm (e.g., 2, 2.5, 3,3.5, or 4 mS/cm) prior to loading to the cation-exchange chromatographycolumn (e.g., SP column). In some embodiments, the conductivity isadjusted by diluting the eluate from the anion-exchange chromatographycolumn with H₂O at about 1-2:1 (e.g., 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1,1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1) ratio. In some embodiments,the conductivity is adjusted by diafiltration. In some embodiments, thecation-exchange chromatography column is run at a pH of about 5.0-6.5(e.g., about 5.0, 5.5, 6.0 or 6.5). In some embodiments, thecation-exchange chromatography column is run with a buffer comprisingphosphate (e.g., NaPO4) concentration ranging from about 0.01 M to about0.1 M (e.g., about 0.01 M, 0.02 M, 0.03 M, 0.04 M, 0.05 M, 0.06 M, 0.07M, 0.08 M, 0.09 M, or 0.1 M). In some embodiments, a suitable pH isabout 5.0-6.5 (e.g., about 5.0, 5.5, 6.0, or 6.5).

In some embodiments, an impure preparation or an intermediate eluate orflow-through is adjusted to phosphate (e.g., NaPO4) concentrationranging from about 0.001 M to about 0.01 M (e.g., about 0.001 M, 0.002M, 0.003 M, 0.004 M, 0.005 M, 0.006 M, 0.007 M, 0.008 M, 0.009 M, or0.01 M) and pH of about 5.0-6.5 (e.g., about 5.0, 5.5, 6.0, or 6.5)prior to loading the mixed-mode chromatography column (e.g., HA column).In some embodiments, the mixed-mode chromatography column (e.g., HAcolumn), once loaded, is washed with a wash buffer containing phosphate(e.g., 1-10 mM sodium or potassium phosphate) at or near neutral pH. Insome embodiments, the loaded mixed-mode chromatography column (e.g., HAcolumn) is washed with a wash buffer having a phosphate concentrationranging from about 10-20 mM (e.g., about 10-18 mM, 10-16 mM, 10-15 mM,12-20 mM, 14-18 mM, 14-16 mM). In some embodiments, the loadedmixed-mode chromatography column (e.g., HA column) is washed with a washbuffer having a phosphate concentration of or greater than 10 mM, 11 mM,12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM. In someembodiments, elution from a mixed-mode chromatography column (e.g., HAcolumn) is achieved with a gradient phosphate buffer. In someembodiments, a suitable elution buffer may have a phosphate gradient ofapproximately 1-400 mM (e.g., 1-300 mM, 1-200 mM, 1-150 mM, 1-100 mM,10-350 mM, 10-300 mM, 10-250 mM, 10-200 mM, 10-150 mM, 10-140 mM, 10-130mM, 10-120 mM, 10-110 mM, 10-100 mM, 10-90 mM, 10-80 mM, 10-70 mM, 10-60mM, 10-50 mM) sodium phosphate or potassium phosphate. In someembodiments, elution from an HA column is achieved by stepwiseincreasing the phosphate concentration in the elution buffer. In someembodiments, stepwise elution buffers may have a phosphate (e.g., sodiumphosphate) concentration selected from 10 mM, 20 mM, 30 mM, 40 mM, 50mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM,150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM. In some embodiments,elution from a mixed-mode chromatography column (e.g., HA column) isachieved by an elution buffer having a phosphate (e.g., sodiumphosphate) concentration ranging from about 50 mM to 150 mM (e.g.,selected from the phosphate (e.g., sodium phosphate) concentration of 50mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM,150 mM, and combination thereof).

In some embodiments, an impure preparation or an intermediate eluate orflow-through is adjusted to salt (e.g., NaCl) concentration ranging fromabout 0.5 M to about 2.0 M (e.g., about 0.5 M, 1.0 M, 1.1 M, 1.2 M, 1.3M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, or 2.0 M NaCl) at pH ofabout 4.5-6.0 (e.g., about 4.5, 5.0, 5.5, or 6.0) prior to loading ontothe hydrophobic interaction chromatography column (e.g., phenyl column).In some embodiments, the hydrophobic interaction chromatography column,once loaded, is washed using a wash buffer comprising salt (e.g., NaCl)concentration ranging from about 0.5 M to 2.0 M (e.g., about 0.5 M, 1.0M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, or 2.0M NaCl) at pH of about 4.5-6.0 (e.g., about 4.5, 5.0, 5.5, or 6.0). Insome embodiments, the hydrophobic interaction chromatography column iseluted using a elution buffer comprising salt (e.g., NaCl) concentrationranging from about 0.1 M to about 0.5 M (e.g., about 0.1 M, 0.2 M, 0.3M, 0.4 M, or 0.5 M NaCl) at pH of about 4.5-6.0 (e.g., about 4.5, 5.0,5.5, or 6.0).

In some embodiments, each of the anion-exchange chromatography,cation-exchange chromatography, mixed-mode chromatography, andhydrophobic interaction chromatography column has a height ranging from14-25 cm (e.g., 15-25 cm, 15-20 cm, 14-24 cm, 14-22 cm, 14-20 cm, or16-18 cm). In some embodiments, each of the anion-exchangechromatography, cation-exchange chromatography, mixed-modechromatography, and hydrophobic interaction chromatography column has aheight of approximately 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25 cm.

In some embodiments, an inventive method according to the presentinvention includes a step of viral inactivation before loading theimpure preparation onto the first chromatography column. In someembodiments, the step of viral inactivation includes adding a detergentto the impure preparation. In some embodiments, an inventive methodaccording to the invention further includes a step of viral removalafter the last Chromatography column. In some embodiments, a method ofthe invention further includes a step of ultrafiltration and/ordiafiltration. In some embodiments, the step ofultrafiltration and/ordiafiltration includes exchanging the purified recombinant I2S proteininto a drug formulation buffer.

In some embodiments, the present invention is used to purify arecombinant I2S protein having an amino acid sequence at least about 50%(e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%) identical to SEQ ID NO: 1. In some embodiments, thepresent invention is used to purify a recombinant I2S protein having anamino acid sequence identical to SEQ ID NO: 1.

In some embodiments, the present invention is used to purify arecombinant I2S protein produced by mammalian cells cultured insuspension in a serum-free medium. In some embodiments, a serum-freemedium suitable for the invention lacks animal-derived components.

In some embodiments, a serum-free medium suitable for the invention is achemically-defined medium. In some embodiments, the mammalian cells arecultured in a bioreactor. In some embodiments, the mammalian cellsco-express the recombinant I2S protein and formylglycine generatingenzyme (FGE). In some embodiments, the mammalian cells are human cells.

In some embodiments, an impure preparation used in a method of theinvention is prepared from the serum-free medium containing recombinantI2S protein secreted from the mammalian cells. In some embodiments, animpure preparation used in a method of the invention is thawed from afrozen medium preparation.

In some embodiments, a purified recombinant I2S protein according to thepresent invention contains, on average, 16-22 (e.g., 16-21, 16-20,16-19, 17-22, 17-21, 17-20, 17-19) sialic acids per molecule. In someembodiments, a purified recombinant I2S protein according to the presentinvention contains, on average, 16, 17, 18, 19, 20, 21, or 22 sialicacids per molecule.

In some embodiments, a purified recombinant I2S protein according to thepresent invention has at least about 70% (e.g., at least about 77%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%) conversion of the cysteine residuecorresponding to Cys59 of human I2S (SEQ ID NO:1) to C_(α)-formylglycine(FGly). In some embodiments, a purified recombinant I2S proteinaccording to the present invention has substantially 100% conversion ofthe cysteine residue corresponding to Cys59 of human I2S (SEQ ID NO: 1)to C_(α)-formylglycine (FGly). In some embodiments, a purifiedrecombinant I2S protein according to the present invention has specificactivity of at least 20 U/mg, 30 U/mg, 40 U/mg, 50 U/mg, 60 U/mg, 70U/mg, 80 U/mg, 90 U/mg, or 100 U/mg as determined by an in vitro sulfaterelease activity assay using heparin disaccharide as substrate.

In some embodiments, a purified recombinant I2S protein according to thepresent invention is characterized with cellular uptake of greater than70%, 75%, 80%, 85%, 90%, 95%, as determined by an in vitro uptake assay.

In some embodiments, a purified recombinant I2S protein according to thepresent invention is characterized with a glycan map comprising sevenpeak groups indicative of neutral (peak group 1), mono-sialylated (peakgroup 2), di-sialylated (peak group 3), monophosphorylated (peak group4), tri-sialylated (peak group 5), tetra-sialylated (peak group 6), anddiphosphorylated (peak group 7) I2S protein, respectively. In someembodiments, the glycan map is generated following a neuraminidasedigestion. In other embodiments, the glycan map is generated followingan alkaline phosphatase digestion.

Among other things, the present invention provides purified recombinantI2S protein as described herein, and pharmaceutical compositions orformulation containing the same. In some embodiments, a formulation isformulated for intravenous, subcutaneous and/or intrathecaladministration. The present invention also provides methods of treatingHunter syndrome by administering into a subject in need of treatment apurified recombinant I2S, pharmaceutical composition or formulationcontaining the same.

As used herein, the terms “I2S protein,” “I2S,” “I2S enzyme,” orgrammatical equivalents, refer to a preparation of recombinant I2Sprotein molecules unless otherwise specifically indicated.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present invention areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present invention, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the invention will become apparent to those skilled in the art fromthe detailed description.

BRIEF DESCRIPTION OF THE DRAWING

The Figures described below, that together make up the Drawing, are forillustration purposes only, not for limitation.

FIG. 1 depicts an exemplary purification scheme for recombinant I2Sproduced in serum free medium.

FIG. 2 depicts an exemplary peptide maps of purified recombinant I2S AFas compared to a reference I2S.

FIG. 3 depicts an exemplary SDS-PAGE (Silver) analysis of purifiedrecombinant I2S AF.

FIG. 4 depicts an exemplary charge profile analysis of purifiedrecombinant I2S AF assessed by ion-exchange chromatography.

FIG. 5 depicts exemplary glycan map profiles of purified recombinant I2SAF.

FIG. 6 depicts an exemplary analysis of activity (U/mg) after a viralinactivation UPB step of a clarified harvest of recombinant I2S.

FIG. 7 depicts an exemplary analysis of SEC-HPLC after a viralinactivation UPB step of a clarified harvest of recombinant I2S.

FIG. 8 depicts exemplary SDS-PAGE treated with silver stain of purifiedrecombinant I2S protein.

FIG. 9 shows an exemplary peptide map for a purified recombinant I2Senzyme produced from the I2S-AF 2D cell line grown under serum-freeculture conditions (top panel) as compared to a reference.

FIG. 10 depicts exemplary glycan profiles generated for purifiedrecombinant I2S enzymes produced using the I2S-AF 2D and 4D cell linesgrown under serum-free cell culture conditions as compared to areference.

FIG. 11 depicts an exemplary charge profile generated for purifiedrecombinant I2S enzyme produced using the I2S-AF 2D cell line grownunder serum-free cell culture conditions as compared to an I2S referencecontrol.

FIG. 12 depicts Table 3 which shows exemplary steps of purificationprocess.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system (e.g., cell culture, organism, etc.). For instance, asubstance that, when administered to an organism, has a biologicaleffect on that organism, is considered to be biologically active.Biological activity can also be determined by in vitro assays (forexample, in vitro enzymatic assays such as sulfate release assays). Inparticular embodiments, where a protein or polypeptide is biologicallyactive, a portion of that protein or polypeptide that shares at leastone biological activity of the protein or polypeptide is typicallyreferred to as a “biologically active” portion. In some embodiments, aprotein is produced and/or purified from a cell culture system, whichdisplays biologically activity when administered to a subject. In someembodiments, a protein requires further processing in order to becomebiologically active. In some embodiments, a protein requiresposttranslational modification such as, but is not limited to,glycosylation (e.g., sialylation), farnesylation, cleavage, folding,formylglycine conversion and combinations thereof, in order to becomebiologically active. In some embodiments, a protein produced as aproform (i.e. immature form), may require additional modification tobecome biologically active.

Cation-independent mannose-6-phosphate receptor (CI-MPR): As usedherein, the term “cation-independent mannose-6-phosphate receptor(CI-MPR)” refers to a cellular receptor that binds mannose-6-phosphate(M6P) tags on acid hydrolase precursors in the Golgi apparatus that aredestined for transport to the lysosome. In addition tomannose-6-phosphates, the CI-MPR also binds other proteins includingIGF-II. The CI-MPR is also known as “M6P/IGF-II receptor,”“CI-MPR/IGF-II receptor,” “IGF-II receptor” or “IGF2 Receptor.” Theseterms and abbreviations thereof are used interchangeably herein.

Chromatography: As used herein, the term “chromatography” refers to atechnique for separation of mixtures. Typically, the mixture isdissolved in a fluid called the “mobile phase,” which carries it througha structure holding another material called the “stationary phase.”Column chromatography is a separation technique in which the stationarybed is within a tube, i.e., column.

Diluent: As used herein, the term “diluent” refers to a pharmaceuticallyacceptable (e.g., safe and non-toxic for administration to a human)diluting substance useful for the preparation of a reconstitutedformulation. Exemplary diluents include sterile water, bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution.

Elution: As used herein, the term “elution” refers to the process ofextracting one material from another by washing with a solvent. Forexample, in ion-exchange chromatography, elution is a process to washloaded resins to remove captured ions.

Eluate: As used herein, the term “eluate” refers to a combination ofmobile phase “carrier” and the analyte material that emerge from thechromatography, typically as a result of eluting.

Enzyme replacement therapy (ERT): As used herein, the term “enzymereplacement therapy (ERT)” refers to any therapeutic strategy thatcorrects an enzyme deficiency by providing the missing enzyme. Onceadministered, enzyme is taken up by cells and transported to thelysosome, where the enzyme acts to eliminate material that hasaccumulated in the lysosomes due to the enzyme deficiency. Typically,for lysosomal enzyme replacement therapy to be effective, thetherapeutic enzyme is delivered to lysosomes in the appropriate cells intarget tissues where the storage defect is manifest.

Equilibrate or Equilibration: As used herein, the terms “equilibrate” or“equilibration” in relation to chromatography refer to the process ofbringing a first liquid (e.g., buffer) into balance with another,generally to achieve a stable and equal distribution of components ofthe liquid (e.g., buffer). For example, in some embodiments, achromatographic column may be equilibrated by passing one or more columnvolumes of a desired liquid (e.g., buffer) through the column.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control individual (or multiple controlindividuals) in the absence of the treatment described herein. A“control individual” is an individual afflicted with the same form oflysosomal storage disease as the individual being treated, who is aboutthe same age as the individual being treated (to ensure that the stagesof the disease in the treated individual and the control individual(s)are comparable).

Impurities: As used herein, the term “impurities” refers to substancesinside a confined amount of liquid, gas, or solid, which differ from thechemical composition of the target material or compound. Impurities arealso referred to as contaminants.

Linker: As used herein, the term “linker” refers to, in a fusionprotein, an amino acid sequence other than that appearing at aparticular position in the natural protein and is generally designed tobe flexible or to interpose a structure, such as an a-helix, between twoprotein moieties. A linker is also referred to as a spacer.

Load: As used herein, the term “load” refers to, in chromatography,adding a sample-containing liquid or solid to a column. In someembodiments, particular components of the sample loaded onto the columnare then captured as the loaded sample passes through the column. Insome embodiments, particular components of the sample loaded onto thecolumn are not captured by, or “flow through”, the column as the loadedsample passes through the column.

Polypeptide: As used herein, a “polypeptide”, generally speaking, is astring of at least two amino acids attached to one another by a peptidebond. In some embodiments, a polypeptide may include at least 3-5 aminoacids, each of which is attached to others by way of at least onepeptide bond. Those of ordinary skill in the art will appreciate thatpolypeptides sometimes include “non-natural” amino acids or otherentities that nonetheless are capable of integrating into a polypeptidechain, optionally.

Pool: As used herein, the term “pool” in relation to chromatographyrefers to combining one or more fractions of fluid that has passedthrough a column together. For example, in some embodiments, one or morefractions which contain a desired component of a sample that has beenseparated by chromatography (e.g., “peak fractions”) can be “pooled”together generate a single “pooled” fraction.

Replacement enzyme: As used herein, the term “replacement enzyme” refersto any enzyme that can act to replace at least in part the deficient ormissing enzyme in a disease to be treated. In some embodiments, the term“replacement enzyme” refers to any enzyme that can act to replace atleast in part the deficient or missing lysosomal enzyme in a lysosomalstorage disease to be treated. In some embodiments, a replacement enzymeis capable of reducing accumulated materials in mammalian lysosomes orthat can rescue or ameliorate one or more lysosomal storage diseasesymptoms. Replacement enzymes suitable for the invention include bothwild-type or modified lysosomal enzymes and can be produced usingrecombinant and synthetic methods or purified from nature sources. Areplacement enzyme can be a recombinant, synthetic, gene-activated ornatural enzyme.

Soluble: As used herein, the term “soluble” refers to the ability of atherapeutic agent to form a homogenous solution. In some embodiments,the solubility of the therapeutic agent in the solution into which it isadministered and by which it is transported to the target site of actionis sufficient to permit the delivery of a therapeutically effectiveamount of the therapeutic agent to the targeted site of action. Severalfactors can impact the solubility of the therapeutic agents. Forexample, relevant factors which may impact protein solubility includeionic strength, amino acid sequence and the presence of otherco-solubilizing agents or salts (e.g., calcium salts). In someembodiments, therapeutic agents in accordance with the present inventionare soluble in its corresponding pharmaceutical composition.

Stability: As used herein, the term “stable” refers to the ability ofthe therapeutic agent (e.g., a recombinant enzyme) to maintain itstherapeutic efficacy (e.g., all or the majority of its intendedbiological activity and/or physiochemical integrity) over extendedperiods of time. The stability of a therapeutic agent, and thecapability of the pharmaceutical composition to maintain stability ofsuch therapeutic agent, may be assessed over extended periods of time(e.g., for at least 1, 3, 6, 12, 18, 24, 30, 36 months or more). In thecontext of a formulation a stable formulation is one in which thetherapeutic agent therein essentially retains its physical and/orchemical integrity and biological activity upon storage and duringprocesses (such as freeze/thaw, mechanical mixing and lyophilization).For protein stability, it can be measure by formation of high molecularweight (HMW) aggregates, loss of enzyme activity, generation of peptidefragments and shift of charge profiles.

Viral Processing: As used herein, the term “viral processing” refers to“viral removal,” in which viruses are simply removed from the sample, or“viral inactivation,” in which the viruses remain in a sample but in anon-infective form. In some embodiments, viral removal may utilizenanofiltration and/or chromatographic techniques, among others. In someembodiments, viral inactivation may utilize solvent inactivation,detergent inactivation, pasteurization, acidic pH inactivation, and/orultraviolet inactivation, among others.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, among other things, an improved methodfor purifying recombinant I2S protein for enzyme replacement therapybased on a process involving less than 6 chromatography steps. In someembodiments, the present invention provides a method of purifyingrecombinant I2S protein from an impure preparation using a process basedon one or more of anion-exchange chromatography, cation-exchangechromatography, mixed-mode chromatography, and hydrophobic interactionchromatography. In some embodiments, the present invention provides amethod of purifying recombinant I2S protein from an impure preparationby conducting Q chromatography, hydroxyapatite (HA) chromatography, SPchromatography, and phenyl chromatography. The present invention furtherprovides purified recombinant I2S protein and method of use.

Various aspects of the invention are described in further detail in thefollowing subsections. The use of subsections is not meant to limit theinvention. Each subsection may apply to any aspect of the invention. Inthis application, the use of“or” means “and/or” unless stated otherwise.

Recombinant I2S Protein

As used herein, an I2S protein is any protein or a portion of a proteinthat can substitute for at least partial activity of naturally-occurringIduronate-2-sulfatase (I2S) protein or rescue one or more phenotypes orsymptoms associated with I2S-deficiency. As used herein, the terms “anI2S enzyme” and “an I2S protein”, and grammatical equivalents, are usedinterchangeably.

Typically, the human I2S protein is produced as a precursor form. Theprecursor form of human I2S contains a signal peptide (amino acidresidues 1-25 of the full length precursor), a pro-peptide (amino acidresidues 26-33 of the full length precursor), and a chain (residues34-550 of the full length precursor) that may be further processed intothe 42 kDa chain (residues 34-455 of the full length precursor) and the14 kDa chain (residues 446-550 of the full length precursor). Typically,the precursor form is also referred to as full-length precursor orfull-length I2S protein, which contains 550 amino acids. The amino acidsequences of the mature form (SEQ ID NO:1) having the signal peptideremoved and full-length precursor (SEQ ID NO:2) of a typical wild-typeor naturally-occurring human I2S protein are shown in Table 1. Thesignal peptide is underlined. In addition, the amino acid sequences ofhuman I2S protein isoform a and b precursor are also provided in Table1, SEQ ID NO:3 and 4, respectively.

TABLE 1 Human Iduronate-2-sulfatase Mature FormSETQANSTTDALNVLLIIVDDLRPSLGCYGDKL VRSPNIDQLASHSLLFQNAFAQQAVCAPSRVSFLTGRRPDTTRLYDFNSYWRVHAGNFSTIPQYFK ENGYVTMSVGKVFHPGISSNHTDDSPYSWSFPPYHPSSEKYENTKTCRGPDGELHANLLCPVDVLD VPEGTLPDKQSTEQAIQLLEKMKTSASPFFLAVGYHKPHIPFRYPKEFQKLYPLENITLAPDPEVP DGLPPVAYNPWMDIRQREDVQALNISVPYGPIPVDFQRKIRQSYFASVSYLDTQVGRLLSALDDLQ LANSTIIAFTSDHGWALGEHGEWAKYSNFDVATHVPLIFYVPGRTASLPEAGEKLFPYLDPFDSAS QLMEPGRQSMDLVELVSLFPTLAGLAGLQVPPRCPVPSFHVELCREGKNLLKHFRFRDLEEDPYLP GNPRELIAYSQYPRPSDIPQWNSDKPSLKDIKIMGYSIRTIDYRYTVWVGFNPDEFLANFSDIHAG ELYFVDSDPLQDHNMYNDSQGGDLFQLLMP(SEQ ID NO: 1) Full-Length MPPPRTGRGLLWLGLVLSSVCVALGSETQANST PrecursorTDALNVLLIIVDDLRPSLGCYGDKLVRSPNIDQ (Isoform a)LASHSLLFQNAFAQQAVCAPSRVSFLTGRRPDT TRLYDFNSYWRVHAGNFSTIPQYFKENGYVTMSVGKVFHPGISSNHTDDSPYSWSFPPYHPSSEKY ENTKTCRGPDGELHANLLCPVDVLDVPEGTLPDKQSTEQAIQLLEKMKTSASPFFLAVGYHKPHIP FRYPKEFQKLYPLENITLAPDPEVPDGLPPVAYNPWMDIRQREDVQALNISVPYGPIPVDFQRKIR QSYFASVSYLDTQVGRLLSALDDLQLANSTITAFTSDHGWALGEHGEWAKYSNFDVATHVPLIFYV PGRTASLPEAGEKLFPYLDPFDSASQLMEPGRQSMDLVELVSLFPTLAGLAGLQVPPRCPVPSFHV ELCREGKNLLKHFRFRDLEEDPYLPGNPRELIAYSQYPRPSDIPQWNSDKPSLKDIKIMGYSIRTI DYRYTVWVGFNPDEFLANFSDIHAGELYFVDSDPLQDHNMYNDSQGGDLFQLLMP (SEQ ID NO: 2) Isoform bMPPPRTGRGLLWLGLVLSSVCVALGSETQANST PrecursorTDALNVLLIIVDDLRPSLGCYGDKLVRSPNIDQ LASHSLLFQNAFAQQAVCAPSRVSFLTGRRPDTTRLYDFNSYWRVHAGNFSTIPQYFKENGYVTMS VGKVFHPGISSNHTDDSPYSWSFPPYHPSSEKYENTKTCRGPDGELHANLLCPVDVLDVPEGTLPD KQSTEQAIQLLEKMKTSASPFFLAVGYHKPHIPFRYPKEFQKLYPLENITLAPDPEVPDGLPPVAY NPWMDIRQREDVQALNISVPYGPIPVDFQEDQSSTGFRLKTSSTRKYK (SEQ ID NO: 3) Isoform cMPPPRTGRGLLWLGLVLSSVCVALGSETQANST PrecursorTDALNVLLIIVDDLRPSLGCYGDKLVRSPNIDQ LASHSLLFQNAFAQQAVCAPSRVSFLTGRRPDTTRLYDFNSYWRVHAGNFSTIPQYFKENGYVTMS VGKVFHPGISSNHTDDSPYSWSFPPYHPSSEKYENTKTCRGPDGELHANLLCPVDVLDVPEGTLPD KQSTEQAIQLLEKMKTSASPFFLAVGYHKPHIPFRYPKEFQKLYPLENITLAPDPEVPDGLPPVAY NPWMDIRQREDVQALNISVPYGPIPVDFQRKIRQSYFASVSYLDTQVGRLLSALDDLQLANSTIIA FTSDHGFLMRTNT (SEQ ID No: 4)

Thus, in some embodiments, a recombinant I2S protein is mature human I2Sprotein (SEQ ID NO: 1). As disclosed herein, SEQ ID NO: 1 represents thecanonical amino acid sequence for the human I2S protein. In someembodiments, the I2S protein may be a splice isoform and/or variant ofSEQ ID NO: 1, resulting from transcription at an alternative start sitewithin the 5′ UTR of the I2S gene. In some embodiments, a recombinantI2S protein may be a homologue or an analogue of mature human I2Sprotein. For example, a homologue or an analogue of mature human I2Sprotein may be a modified mature human I2S protein containing one ormore amino acid substitutions, deletions, and/or insertions as comparedto a wild-type or naturally-occurring I2S protein (e.g., SEQ ID NO: 1),while retaining substantial I2S protein activity. Thus, in someembodiments, a recombinant I2S protein is substantially homologous tomature human I2S protein (SEQ ID NO: 1). In some embodiments, arecombinant I2S protein has an amino acid sequence at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more homologous to SEQ ID NO: 1. In some embodiments, arecombinant I2S protein is substantially identical to mature human I2Sprotein (SEQ ID NO: 1). In some embodiments, a recombinant I2S proteinhas an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto SEQ ID NO: 1. In some embodiments, a recombinant I2S protein containsa fragment or a portion of mature human I2S protein.

Alternatively, a recombinant I2S protein is full-length I2S protein. Insome embodiments, a recombinant I2S protein may be a homologue or ananalogue of full-length human I2S protein. For example, a homologue oran analogue of full-length human I2S protein may be a modifiedfull-length human I2S protein containing one or more amino acidsubstitutions, deletions, and/or insertions as compared to a wild-typeor naturally-occurring full-length I2S protein (e.g., SEQ ID NO:2),while retaining substantial I2S protein activity. Thus, In someembodiments, a recombinant I2S protein is substantially homologous tofull-length human I2S protein (SEQ ID NO:2). For example, a recombinantI2S protein may have an amino acid sequence at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore homologous to SEQ ID NO:2. In some embodiments, a recombinant I2Sprotein is substantially identical to SEQ ID NO:2. For example, arecombinant I2S protein may have an amino acid sequence at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to SEQ ID NO:2. In some embodiments, arecombinant I2S protein contains a fragment or a portion of full-lengthhuman I2S protein. As used herein, a full-length I2S protein typicallycontains signal peptide sequence.

In some embodiments, a recombinant I2S protein is human I2S isoform aprotein. In some embodiments, a recombinant I2S protein may be ahomologue or an analogue of human I2S isoform a protein. For example, ahomologue or an analogue of human I2S isoform a protein may be amodified human I2S isoform a protein containing one or more amino acidsubstitutions, deletions, and/or insertions as compared to a wild-typeor naturally-occurring human I2S isoform a protein (e.g., SEQ ID NO:3),while retaining substantial I2S protein activity. Thus, in someembodiments, a recombinant I2S protein is substantially homologous tohuman I2S isoform a protein (SEQ ID NO:3). For example, a recombinantI2S protein may have an amino acid sequence at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore homologous to SEQ ID NO:3. In some embodiments, a recombinant I2Sprotein is substantially identical to SEQ ID NO:3. For example, arecombinant I2S protein may have an amino acid sequence at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to SEQ ID NO:3. In some embodiments, arecombinant I2S protein contains a fragment or a portion of human I2Sisoform a protein. As used herein, a human I2S isoform a proteintypically contains a signal peptide sequence.

In some embodiments, a recombinant I2S protein is human I2S isoform bprotein. In some embodiments, a recombinant I2S protein may be ahomologue or an analogue of human I2S isoform b protein. For example, ahomologue or an analogue of human I2S isoform b protein may be amodified human I2S isoform b protein containing one or more amino acidsubstitutions, deletions, and/or insertions as compared to a wild-typeor naturally-occurring human I2S isoform b protein (e.g., SEQ ID NO:4),while retaining substantial I2S protein activity. Thus, in someembodiments, a recombinant I2S protein is substantially homologous tohuman I2S isoform b protein (SEQ ID NO:4). For example, a recombinantI2S protein may have an amino acid sequence at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore homologous to SEQ ID NO:4. In some embodiments, a recombinant I2Sprotein is substantially identical to SEQ ID NO:4. For example, arecombinant I2S protein may have an amino acid sequence at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to SEQ ID NO:4. In some embodiments, arecombinant I2S protein contains a fragment or a portion of human I2Sisoform b protein. As used herein, a human I2S isoform b proteintypically contains a signal peptide sequence.

Homologues or analogues of human I2S proteins can be prepared accordingto methods for altering polypeptide sequence known to one of ordinaryskill in the art such as are found in references that compile suchmethods. In some embodiments, conservative substitutions of amino acidsinclude substitutions made among amino acids within the followinggroups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T;(f) Q, N; and (g) E, D. In some embodiments, a “conservative amino acidsubstitution” refers to an amino acid substitution that does not alterthe relative charge or size characteristics of the protein in which theamino acid substitution is made.

In some embodiments, recombinant I2S proteins may contain a moiety thatbinds to a receptor on the surface of target cells to facilitatecellular uptake and/or lysosomal targeting. For example, such a receptormay be the cation-independent mannose-6-phosphate receptor (CI-MPR)which binds the mannose-6-phosphate (M6P) residues. In addition, theCI-MPR also binds other proteins including IGF-II. In some embodiments,a recombinant I2S protein contains M6P residues on the surface of theprotein. In particular, a recombinant I2S protein may containbis-phosphorylated oligosaccharides which have higher binding affinityto the CI-MPR. In some embodiments, a suitable enzyme contains up toabout an average of about at least 20% bis-phosphorylatedoligosaccharides per enzyme. In other embodiments, a suitable enzyme maycontain about 10%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%bis-phosphorylated oligosaccharides per enzyme.

In some embodiments, recombinant I2S enzymes may be fused to a lysosomaltargeting moiety that is capable of binding to a receptor on the surfaceof target cells. A suitable lysosomal targeting moiety can be IGF-I,IGF-II, RAP, p97, and variants, homologues or fragments thereof (e.g.,including those peptide having a sequence at least 70%, 75%, 80%, 85%,90%, or 95% identical to a wild-type mature human IGF-I, IGF-II, RAP,p97 peptide sequence).

The lysosomal targeting moiety may be conjugated or fused to an I2Sprotein or enzyme at the N-terminus, C-terminus or internally.

Production of Recombinant I2S Proteins

The present invention may be used to purify a recombinant I2S proteinproduced by various means. For example, an I2S protein may berecombinantly produced by utilizing a host cell system engineered toexpress an I2S-encoding nucleic acid. Alternatively, an I2S protein maybe produced by activating an endogenous I2S gene.

It is contemplated that the present invention can be used to purify arecombinant I2S protein produced using various expression system.Suitable expression systems include, for example, egg, baculovirus,plant, yeast, or mammalian cells.

In some embodiments, I2S enzymes are produced in mammalian cells.Non-limiting examples of mammalian cells that may be used in accordancewith the present invention include BALB/c mouse myeloma line (NSO/l,ECACC No: 85110503); human retinoblasts (PER.C6, CruCell, Leiden, TheNetherlands); monkey kidney CV1 line transformed by SV40 (COS-7, ATCCCRL 1651); human embryonic kidney line (HEK293 or 293 cells subclonedfor growth in suspension culture, Graham et al., J. Gen Virol., 36:59,1977); human fibrosarcoma cell line (e.g., HT1080); baby hamster kidneycells (BHK21, ATCC CCL 10); Chinese hamster ovary cells+/−DHFR (CHO,Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); mousesertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980); monkeykidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCCCCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68,1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

In some embodiments, inventive methods according to the presentinvention are used to purify recombinant I2S enzymes produced from humancells (e.g., HT1080). In some embodiments, inventive methods accordingto the present invention are used to purify recombinant I2S enzymesproduced from CHO cells.

Typically, cells that are engineered to express recombinant I2S maycomprise a transgene that encodes a recombinant I2S protein describedherein. It should be appreciated that the nucleic acids encodingrecombinant I2S may contain regulatory sequences, gene controlsequences, promoters, non-coding sequences and/or other appropriatesequences for expressing the recombinant I2S. Typically, the codingregion is operably linked with one or more of these nucleic acidcomponents.

“Regulatory sequences” typically refer to nucleotide sequences locatedupstream (5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, and polyadenylation recognition sequences.Sometimes, “regulatory sequences” are also referred to as “gene controlsequences.”

“Promoter” typically refers to a nucleotide sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Ingeneral, a coding sequence is located 3′ to a promoter sequence. Thepromoter sequence consists of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a nucleotide sequence that can stimulatepromoter activity and may be an innate element of the promoter or aheterologous element inserted to enhance the level or tissue-specificityof a promoter. Promoters may be derived in their entirety from a nativegene, or be composed of different elements derived from differentpromoters found in nature, or even comprise synthetic nucleotidesegments. It is understood by those skilled in the art that differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions.

The “3′ non-coding sequences” typically refer to nucleotide sequenceslocated downstream of a coding sequence and include polyadenylationrecognition sequences and other sequences encoding regulatory signalscapable of affecting mRNA processing or gene expression. Thepolyadenylation signal is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor.

The “translation leader sequence” or “5′ non-coding sequences” typicallyrefers to a nucleotide sequence located between the promoter sequence ofa gene and the coding sequence. The translation leader sequence ispresent in the fully processed mRNA upstream of the translation startsequence. The translation leader sequence may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency.

Typically, the term “operatively linked” or “operably linked” refers tothe association of two or more nucleic acid fragments on a singlenucleic acid fragment so that the function of one is affected by theother. For example, a promoter is operatively linked with a codingsequence when it is capable of affecting the expression of that codingsequence (i.e., that the coding sequence is under the transcriptionalcontrol of the promoter). Coding sequences can be operatively linked toregulatory sequences in sense or antisense orientation.

The coding region of a transgene may include one or more silentmutations to optimize codon usage for a particular cell type. Forexample, the codons of an I2S transgene may be optimized for expressionin a vertebrate cell. In some embodiments, the codons of an I2Stransgene may be optimized for expression in a mammalian cell. In someembodiments, the codons of an I2S transgene may be optimized forexpression in a human cell.

Optionally, a construct may contain additional components such as one ormore of the following: a splice site, an enhancer sequence, a selectablemarker gene under the control of an appropriate promoter, an amplifiablemarker gene under the control of an appropriate promoter, and a matrixattachment region (MAR) or other element known in the art that enhancesexpression of the region where it is inserted.

Once transfected or transduced into host cells, a suitable vector canexpress extrachromosomally (episomally) or integrate into the hostcell's genome.

Activation of Recombinant I2S Proteins

Typically, a recombinant I2S enzyme is activated by thepost-translational modification of a conserved cysteine (correspondingto amino acid 59 of mature human I2S) to formylglycine, also known as2-amino-3-oxopropionic acid, or oxo-alanine. Such post-translationalmodification can be carried out by an enzyme known as FormylglycineGenerating Enzyme (FGE). Thus, in some embodiments, recombinant I2Senzymes are produced in cells that also express FGE protein. Inparticular embodiments, recombinant I2S enzymes are produced in cellsthat have increased or enhanced expression of FGE protein. For example,cells may be engineered to over-express FGE in combination withrecombinant I2S to facilitate the production of I2S preparations havinghigh levels of active enzyme. In some embodiments, over-expression ofFGE is achieved by expression (e.g., over-expression) of an exogenousFGE using standard recombinant technology. In some embodiments,over-expression of FGE is achieved by activated or enhanced expressionof an endogenous FGE by, for example, activating or enhancing thepromoter of the endogenous FGE gene. In some cases, the nucleic acidencoding recombinant I2S and the nucleic acid encoding a recombinant FGEprotein are linked by a nucleic acid (e.g., a spacer sequence) having asequence corresponding to an internal ribosomal entry site.

Any FGE having ability to convert cysteine to formylglycine may be usedin the present invention. Exemplary nucleic acid and amino acidsequences for FGE proteins are disclosed in US 2004-0229250, the entirecontents relating to such sequences and the sequences themselves areincorporated herein by reference in their entireties. It should beappreciated that the nucleic acids encoding recombinant FGE may compriseregulatory sequences, gene control sequences, promoters, non-codingsequences and/or other appropriate sequences for expressing the FGE.Typically, the coding region is operably linked with one or more ofthese nucleic acid components.

Cell Culture Medium and Condition

Various cell culture medium and conditions may be used to produce arecombinant I2S protein. For example, a recombinant I2S protein may beproduced in serum-containing or serum-free medium. In some embodiments,a recombinant I2S protein is produced in serum-free medium. In someembodiments, a recombinant I2S protein is produced in an animal freemedium, i.e., a medium that lacks animal-derived components. In someembodiments, a recombinant I2S protein is produced in a chemicallydefined medium. As used herein, the term “chemically-defined nutrientmedium” refers to a medium of which substantially all of the chemicalcomponents are known. In some embodiments, a chemically defined nutrientmedium is free of animal-derived components such as serum, serum derivedproteins (e.g., albumin or fetuin), and other components. In some cases,a chemically-defined medium comprises one or more proteins (e.g.,protein growth factors or cytokines.) In some cases, achemically-defined nutrient medium comprises one or more proteinhydrolysates. In other cases, a chemically-defined nutrient medium is aprotein-free media, i.e., a serum-free media that contains no proteins,hydrolysates or components of unknown composition.

In some embodiments, a chemically defined medium may be supplemented byone or more animal derived components. Such animal derived componentsinclude, but are not limited to, fetal calf serum, horse serum, goatserum, donkey serum, human serum, and serum derived proteins such asalbumins (e.g., bovine serum albumin or human serum albumin).

Various cell culture conditions may be used to produce recombinant I2Sproteins at large scale including, but not limited to, roller bottlecultures, bioreactor batch cultures and bioreactor fed-batch cultures.In some embodiments, recombinant I2S protein is produced by cellscultured in suspense. In some embodiments, recombinant I2S protein isproduced by adherent cells.

Exemplary cell media and culture conditions are described in theExamples sections. Additional exemplary methods and compositions forproducing recombinant I2S protein are described in the provisionalapplication entitled “Methods and Compositions for Producing RecombinantIduronate-2-Sulfatase” filed herewith on even date, the entiredisclosure of which is hereby incorporated by reference.

Purification of Recombinant I2S Protein

In some embodiments, the present invention provides a method ofpurifying recombinant I2S protein from an impure preparation using aprocess based on one or more of anion-exchange chromatography,cation-exchange chromatography, mixed-mode chromatography, andhydrophobic interaction chromatography. In some embodiments, aninventive method according to the present invention involves less than 6(e.g., less than 5, less than 4, or less than 3) chromatography steps.In some embodiments, an inventive method according to the presentinvention involves 2, 3, 4 or 5 chromatography steps. In someembodiments, an inventive method according to the present inventioninvolves 4 chromatography steps. In some embodiments, an inventivemethod according to the present invention conducts anion-exchangechromatography, mixed-mode chromatography, cation-exchangechromatography, and hydrophobic interaction chromatography in thatorder.

Impure Preparation

As used herein, an impure preparation can be any biological materialincluding unprocessed biological material containing recombinant I2Sprotein. For example, an impure preparation may be unprocessed cellculture medium containing recombinant I2S protein secreted from thecells (e.g., mammalian cells) producing I2S protein or raw cell lysatescontaining I2S protein. In some embodiments, an impure preparation maybe partially processed cell medium or cell lysates. For example, cellmedium or cell lysates can be concentrated, diluted, treated with viralinactivation, viral processing or viral removal. In some embodiments,viral removal may utilize nanofiltration and/or chromatographictechniques, among others. In some embodiments, viral inactivation mayutilize solvent inactivation, detergent inactivation, pasteurization,acidic pH inactivation, and/or ultraviolet inactivation, among others.Cell medium or cell lysates may also be treated with protease, DNases,and/or RNases to reduce the level of host cell protein and/or nucleicacids (e.g., DNA or RNA). In some embodiments, unprocessed or partiallyprocessed biological materials (e.g., cell medium or cell lysate) may befrozen and stored at a desired temperature (e.g., 2-8° C., −4° C., −25°C., −75° C.) for a period time and then thawed for purification. As usedherein, an impure preparation is also referred to as starting materialor loading material.

Anion-Exchange Chromatography

In some embodiments, provided methods for purifying recombinant I2Sinclude anion-exchange chromatography. In brief, anion exchangechromatography is a chromatographic technique which relies oncharge-charge interactions between a negatively charged compound and apositively charged resin. In some embodiments, the anion-exchangechromatography is strong anion-exchange chromatography. In someembodiments, anion-exchange chromatography is employed as a firstpurification step for a therapeutic protein (e.g., recombinant I2S).

Exemplary anion exchange resins include, but are not limited to,quaternary amine resins or “Q-resins” (e.g., Capto™-Q, Q-Sepharose®, QAESephadex®); diethylaminoethane (DEAE) resins (e.g., DEAE-Trisacryl®,DEAE Sepharose®, benzoylated naphthoylated DEAE, diethylaminoethylSephacel®); Amberjet® resins; Amberlyst® resins; Amberlite® resins(e.g., Amberlite® IRA-67, Amberlite® strongly basic, Amberlite® weaklybasic), cholestyramine resin, ProPac® resins (e.g., ProPac® SAX-10,ProPac® WAX-10, ProPac® WCX-10); TSK-GEL resins (e.g., TSKgel DEAE-NPR;TSKgel DEAE-5PW); and Acclaim® resins. In certain embodiments, the anionexchange resin is a Q resin.

Typical mobile phases for anionic exchange chromatography includerelatively polar solutions, such as water, acetonitrile, organicalcohols such as methanol, ethanol, and isopropanol, or solutionscontaining 2-(N-morpholino)-ethanesulfonic acid (MES). Thus, in certainembodiments, the mobile phase includes about 0%, 1%, 2%, 4%, 6%, 8%,10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100% polar solution. Incertain embodiments, the mobile phase comprises between about 1% toabout 100%, about 5% to about 95%, about 10% to about 90%, about 20% toabout 80%, about 30% to about 70%, or about 40% to about 60% polarsolution at any given time during the course of the separation.

Generally, a mobile phase includes a salt. For example, a salt (e.g.,sodium chloride) can elute a bound protein from an anion exchange column(e.g., the counter ion is chloride and it is exchanged for the targetprotein, which is then released). In some embodiments, the mobile phaseincludes a salt concentration between about 0 to about 1.0M, e.g.,between about 0 to about 0.8M, between about 0 to about 0.6M, betweenabout 0 to about 0.5M, between about 0 to about 0.4M, between about0.05M to about 0.50M, between about 0.10M to about 0.45M, between about0.10M to about 0.40M, or between about 0.15M to about 0.40M. In someembodiments, the mobile phase includes a salt concentration ofapproximately 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M,0.09M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, or 1.0M. Insome embodiments, salt concentration in the mobile phase is a gradient(e.g., linear or non-linear gradient). In some embodiments, saltconcentration in the mobile phase is constant. In some embodiments, saltconcentration in the mobile phase may increase or decrease stepwise.

Typically, the mobile phase is buffered. In certain embodiments, themobile phase is not buffered. In certain embodiments, the mobile phaseis buffered to a pH between about 5 to about 14. In certain embodiments,the mobile phase is buffered to a pH between about 5 to about 10. Incertain embodiments, the mobile phase is buffered to a pH between about5 to about 7. In certain embodiments, the mobile phase is buffered to apH of about 6.5. In certain embodiments, the mobile phase is buffered toa pH of about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.

In some embodiments, an impure preparation or an intermediate eluate orflow-through is adjusted to pH of about 5.0, 5.5, 6.0, 6.5, 7.0 or 7.5and the conductivity of about 2 mS/cm, 4 mS/cm, 6 mS/cm, 8 mS/cm, 10mS/cm, 12 mS/cm, 14 mS/cm, 16 mS/cm, 18 mS/cm, or 20 mS/cm prior toloading to the anion-exchange chromatography column (e.g., Q column).

The pH may be adjusted using sodium acetate (e.g., 1M) and theconductivity may be adjusted using sodium chloride (e.g., 5M). Onceloaded, an anion-exchange chromatography column may be washed using awash buffer comprising salt (e.g., NaCl) concentration ranging fromabout 140 mM to about 200 mM (e.g., about 140 mM, 145 mM, 150 mM, 155mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, or200 mM) with pH of about 5.0-7.5 (e.g., about 5.0, 5.5, 6.0, 6.5, 7.0 or7.5). An anion-exchange chromatography column may be eluted using anelution buffer comprising a linear NaCl gradient. A suitable exemplarylinear NaCl gradient may contain a range from about 0-500 mM NaCl (e.g.,about 0-400 mM, about 0-350 mM, about 0-300 mM, about 50-500 mM, about150-500 mM, about 150-450 mM, about 150-400 mM).

Cation Exchange Chromatography

In some embodiments, provided methods for purifying recombinant I2Sinclude cation-exchange chromatography. In brief, cation exchangechromatography is a chromatographic technique which relies oncharge-charge interactions between a positively charged compound and anegatively charged resin. In some embodiments, the cation-exchangechromatography is strong cation-exchange chromatography.

Cation exchange chromatography is generally practiced with either astrong or weak cation exchange column, containing a sulfonium ion, orwith a weak cation exchanger, having usually a carboxymethyl (CM) orcarboxylate (CX) functional group. Many suitable cation exchange resinsare known in the art and are commercially available and include, but arenot limited to SP-Sepharose®, CM Sepharose; Amberjet® resins; Amberlyst®resins; Amberlite® resins (e.g., Amberlite® IRA 120); ProPac® resins(e.g., ProPac® ScX-10, ProPac® WCX-10, ProPac® WCX-10); TSK-GEL resins(e.g., TSKgel BioAssist S; TSKgel SP-2SW, TSKgel SP-SPW; TSKgel SP-NPR;TSKgel SCX; TSKgel SP-STAT; TSKgel CM-5PW; TSKgel OApak-A; TSKgelCM-2SW, TSKgel CM-3SW, and TSKgel CM-STAT); and Acclaim® resins. Incertain embodiments, the anion exchange resin is an SP-Sepharose Resin®.

Typical mobile phases for cationic exchange chromatography includerelatively polar solutions, such as water, acetonitrile, organicalcohols such as methanol, ethanol, and isopropanol, or solutionscontaining 2-(N-morpholino)-ethanesulfonic acid (MES). Thus, in certainembodiments, the mobile phase includes about 0%, 1%, 2%, 4%, 6%, 8%,10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100% polar solution. Incertain embodiments, the mobile phase includes between about 1% to about100%, about 5% to about 95%, about 10% to about 90%, about 20% to about80%, about 30% to about 70%, or about 40% to about 60% polar solution atany given time during the course of the separation.

Generally, a mobile phase includes a salt. For example, a salt (e.g.,sodium chloride, sodium phosphate, etc.) can elute a bound protein froman cation exchange column (e.g., the counter ion is sodium and it isexchanged for the target protein, which is then released). In someembodiments, the mobile phase includes a salt concentration betweenabout 0 to about 1.0M, e.g., between about 0 to about 0.8M, betweenabout 0 to about 0.6M, between about 0 to about 0.5M, between about 0 toabout 0.4M, between about 0.05M to about 0.50M, between about 0.10M toabout 0.45M, between about 0.10M to about 0.40M, or between about 0.15Mto about 0.40M. In some embodiments, the mobile phase includes a saltconcentration of approximately 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M,0.07M, 0.08M, 0.09M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M,0.9M, or 1.0M. In some embodiments, salt concentration in the mobilephase is a gradient (e.g., linear or non-linear gradient). In someembodiments, salt concentration in the mobile phase is constant. In someembodiments, salt concentration in the mobile phase may increase ordecrease stepwise.

Typically, the mobile phase is buffered. In certain embodiments, themobile phase is not buffered. In certain embodiments, the mobile phaseis buffered to a pH between about 5 to about 14. In certain embodiments,the mobile phase is buffered to a pH between about 5 to about 10. Incertain embodiments, the mobile phase is buffered to a pH between about5 to about 7. In certain embodiments, the mobile phase is buffered to apH of about 6.5. In certain embodiments, the mobile phase is buffered toa pH of about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.

In some embodiments, an impure preparation or an intermediate eluate orflow-through is adjusted to conductivity ranging between about 1 mS/cmand 20 mS/cm (e.g., between about 1 mS/cm and 15 mS/cm, between about 1mS/cm and 10 mS/cm, between about 1 mS/cm and 8 mS/cm, between about 1mS/cm and 6 mS/cm, between about 1 mS/cm and 4 mS/cm, between about 2mS/cm and 4 mS/cm) prior to loading to the cation-exchangechromatography column (e.g., SP column). In particular embodiments, animpure preparation or an intermediate eluate or flow-through is adjustedto conductivity ranging between [[n]] about 2 mS/cm and 4 mS/cm (e.g.,2, 2.5, 3, 3.5, or 4 mS/cm) prior to loading to the cation-exchangechromatography column (e.g., SP column). The conductivity may beadjusted by diluting an impure preparation or an intermediate eluate orflow-through with H₂O at, e.g., 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1,2.0:1, 2.5:1, 3.0:1, 4.0:1, 5.0:1, or 10:1 ratio. The conductivity mayalso be adjusted by diafiltration into a desired buffer. In someembodiments, a cation-exchange chromatography column is run at a pH ofabout 5.0-6.5 (e.g., about 5.0, 5.5, 6.0 or 6.5). In some embodiments, acation-exchange chromatography column is run with a buffer comprisingphosphate (e.g., NaPO4) concentration ranging from about 0.01 M to about0.1 M (e.g., about 0.01 M, 0.02 M, 0.03 M, 0.04 M, 0.05 M, 0.06 M, 0.07M, 0.08 M, 0.09 M, or 0.1 M). In some embodiments, a suitable pH isabout 5.0-6.5 (e.g., about 5.0, 5.5, 6.0, or 6.5).

Mixed Mode Chromatography

Hydroxyapatite chromatography (HA) is considered to be a“pseudo-affinity” chromatography or “mixed-mode” ion exchange and may beused in accordance with the present invention. Hydroxyapatite is aunique form of calcium phosphate used in fractionation and purificationof biological molecules. In some cases, crystalline hydroxyapatite maybe used, although the fragility of the crystals may limit flow ratesand/or column longevity. Two types of chemically pure ceramichydroxyapatite, CHT ceramic hydroxyapatite Types I and II aremacroporous, spherical and can be used at high flow rates and pressures.Type I generally has a high protein binding capacity, while Type IIgenerally has a lower binding capacity for proteins. In general, theformula of hydroxyapatite is Ca₁₀(PO₄)₆(OH)₂ (Kawasaki, et al 1985). Thefunctional groups include positively charged pairs of crystal calciumions (C-sites) and clusters of six negatively charged oxygen atomsassociated with triplets of crystal phosphates (P-sites). C-sites,P-sites and hydroxyls are distributed in a fixed pattern on the crystalsurface, generally leading to complex interactions with proteins andother molecules.

A sample may be loaded onto an HA column in low ionic strength phosphatebuffer (e.g., 1-10 mM sodium or potassium phosphate) at or near neutralpH. In some embodiments, an impure preparation or an intermediate eluateor flow-through is adjusted to phosphate (e.g., NaPO4) concentrationranging from about 0.001 M to about 0.01 M (e.g., about 0.001 M, 0.002M, 0.003 M, 0.004 M, 0.005 M, 0.006 M, 0.007 M, 0.008 M, 0.009 M, or0.01 M) and pH of about 5.0-6.5 (e.g., about 5.0, 5.5, 6.0, or 6.5)prior to loading the mixed-mode chromatography column (e.g., HA column).The loaded HA column are typically washed with a wash buffer having aphosphate concentration comparable to that of the loading buffer. Insome embodiments, the mixed-mode chromatography column (e.g., HAcolumn), once loaded, is washed with a wash buffer containing phosphate(e.g., 1-10 mM sodium or potassium phosphate) at or near neutral pH. Forexample, a suitable wash buffer may have a phosphate concentration ofabout 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM. Insome embodiments, it may be desirable to increase the amount ofphosphate in the wash buffer to create a more stringent wash condition.It is contemplated that the M6P levels, in particular di-M6P levels, onthe surface of I2S proteins are important for lysosomal targeting.Increased phosphate concentration in the wash buffer may selectivelyretain I2S proteins with high levels of M6P, in particular, di-M6P onthe HA column. Thus, in some embodiments, a desired wash buffer may havea phosphate concentration of or greater than 10 mM, 11 mM, 12 mM, 13 mM,14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM. In some embodiments,the loaded mixed-mode chromatography column (e.g., HA column) is washedwith a wash buffer having a phosphate concentration ranging from about10-20 mM (e.g., about 10-18 mM, 10-16 mM, 10-15 mM, 12-20 mM, 14-18 mM,14-16 mM). In some embodiments, the loaded mixed-mode chromatographycolumn (e.g., HA column) is washed with a wash buffer having a phosphateconcentration of or greater than 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM.

Elution from an HA column is typically achieved with a gradientphosphate buffer. For example, a suitable elution buffer may have aphosphate gradient of approximately 1-400 mM (e.g., 1-300 mM, 1-200 mM,1-150 mM, 1-100 mM, 10-350 mM, 10-300 mM, 10-250 mM, 10-200 mM, 10-150mM, 10-140 mM, 10-130 mM, 10-120 mM, 10-110 mM, 10-100 mM, 10-90 mM,10-80 mM, 10-70 mM, 10-60 mM, 10-50 mM) sodium phosphate. In someembodiments, elution from an HA column is achieved by stepwiseincreasing the phosphate concentration in the elution buffer. In someembodiments, stepwise elution buffers may have a phosphate concentrationselected from 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 200 mM, 250 mM, 300mM, 350 mM, 400 mM. In some embodiments, elution from a mixed-modechromatography column (e.g., HA column) is achieved by an elution bufferhaving a phosphate (e.g., sodium phosphate) concentration ranging fromabout 50 mM to about 150 mM (e.g., selected from a phosphate (e.g.,sodium phosphate) concentration of about 50 mM, 60 mM, 70 mM, 80 mM, 90mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, and combinationthereof).

It will be appreciated that many different combinations of conditionsfor HA chromatography are known and may be used to adjust the parametersto be suitable for a particular protein of interest (e.g., recombinantI2S).

Hydrophobic Interaction Chromatography

Hydrophobic Interaction Chromatography (HIC) is a separation techniquethat uses the properties of hydrophobicity to separate proteins from oneanother. In this type of chromatography, hydrophobic groups such asphenyl, octyl, or butyl, are attached to the stationary column. Proteinsthat pass through the column that have hydrophobic amino acid sidechains on their surfaces are able to interact with and bind to thehydrophobic groups on the column. HIC columns are known, and include forexample, Phenyl Sepharose.

HIC separations are often designed using the opposite conditions ofthose used in ion exchange chromatography. In general, a buffer with ahigh ionic strength, usually ammonium sulfate, is initially applied tothe column. The salt in the buffer reduces the solvation of samplesolutes thus as solvation decreases, hydrophobic regions that becomeexposed are adsorbed by the medium. The stationary phase is generallydesigned to form hydrophobic interactions with other molecules. Theseinteractions are generally too weak in water, however, addition of salts(e.g., Na₂SO₄, K₂SO₄, (NH₄)₂SO₄, NaCl, NH₄Cl, NaBr, and NaSCN) to thebuffer results in hydrophobic interactions. In some embodiments, themobile phase includes a salt concentration between about 0.1M to about3.0M, e.g., between about 0.1M to about 1.5M, between about 0.2M toabout 0.8M, or between about 0.3M to about 0.5M.

In certain embodiments, the mobile phase is buffered. In certainembodiments, the mobile phase is not buffered. In certain embodiments,the mobile phase is buffered to a pH between about 5 to about 14. Incertain embodiments, the mobile phase is buffered to a pH between about5 to about 10. In certain embodiments, the mobile phase is buffered to apH between about 5 to about 7. In certain embodiments, the mobile phaseis buffered to a pH of about 5.0.

In some embodiments, an impure preparation or an intermediate eluate orflow-through is adjusted to salt (e.g., NaCl) concentration ranging fromabout 0.5 M to about 2.0 M (e.g., about 0.5 M, 1.0 M, 1.1 M, 1.2 M, 1.3M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, or 2.0 M) at pH of about4.5-6.0 (e.g., about 4.5, 5.0, 5.5, or 6.0) prior to loading onto thehydrophobic interaction chromatography column (e.g., phenyl column).Once loaded, a hydrophobic interaction chromatography column may bewashed using a wash buffer comprising salt (e.g., NaCl) concentrationranging from about 0.5 M to about 2.0 M (e.g., about 0.5 M, 1.0 M, 1.1M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, or 2.0 M) atpH of about 4.5-6.0 (e.g., about 4.5, 5.0, 5.5, or 6.0). In someembodiments, the hydrophobic interaction chromatography column is elutedusing a elution buffer comprising salt (e.g., NaCl) concentrationranging from about 0.1 M to about 0.5 M (e.g., about 0.1 M, 0.2 M, 0.3M, 0.4 M, or 0.5 M) at pH of about 4.5-6.0 (e.g., about 4.5, 5.0, 5.5,or 6.0).

Characterization of Purified I2S Proteins

Purified recombinant I2S protein may be characterized using variousmethods.

Purity

The purity of purified recombinant I2S protein is typically measured bythe level of various impurities (e.g., host cell protein or host cellDNA) present in the final product. For example, the level of host cellprotein (HCP) may be measured by ELISA or SDS-PAGE. In some embodiments,the purified recombinant I2S protein contains less than 150 ng HCP/mgI2S protein (e.g., less than 140, 130, 120, 110, 100, 90, 80, 70, 60,50, 40, 30, 30, 20, 10 ng HCP/mg I2S protein). In some embodiments, thepurified recombinant I2S protein, when subject to SDS-PAGE with silverstaining, has no new bands with intensity greater than the 0.05%, 0.01%,0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5% assay control.Various assay controls may be used, in particular, those acceptable toregulatory agencies such as FDA.

Specific Activity

Purified recombinant I2S protein may also be characterized by evaluatingfunctional and/or biological activity. The enzyme activity of arecombinant I2S composition may be determined using methods known in theart. Typically the methods involve detecting the removal of sulfate froma synthetic substrate, which is known as sulphate release assay. Oneexample of an enzyme activity assay involves the use of ionchromatography. This method quantifies the amount of sulfate ions thatare enzymatically released by recombinant I2S from a substrate. Thesubstrate may be a natural substrate or a synthetic substrate. In somecases, the substrate is heparin sulfate (e.g., heparin disaccharide),dermatan sulfate, or a functional equivalent thereof. Typically, thereleased sulfate ion is analyzed by ion chromatography with aconductivity detector. In this example, the results may be expressed asU/mg of protein where 1 Unit is defined as the quantity of enzymerequired to release 1 μmole sulfate ion per hour from the substrate. Insome embodiments, purified recombinant I2S protein has a specificactivity, as measured by in vitro sulfate release activity assay usingheparin disaccharide as substrate, ranging from about 0-100 U/mg, about10-100 U/mg, about 10-80 U/mg, about 20-80 U/mg, about 20-70 U/mg, about20-60 U/mg, about 20-50 U/mg, about 30-100 U/mg, about 30-90 U/mg, about30-80 U/mg, about 30-70 U/mg, about 30-60 U/mg, about 40-100 U/mg, about40-90 U/mg, about 40-80 U/mg, about 40-70 U/mg, about 40-60 U/mg. Insome embodiments, purified recombinant I2S protein has a specificactivity, as measured by in vitro sulfate release activity assay usingheparin disaccharide as substrate, of at least about 5 U/mg, about 10U/mg, about 15 U/mg, about 20 U/mg, about 25 U/mg, about 30 U/mg, about35 U/mg, about 40 U/mg, about 45 U/mg, about 50 U/mg, about 55 U/mg,about 60 U/mg, about 65 U/mg, about 70 U/mg, about 75 U/mg, about 80U/mg, about 85 U/mg, about 90 U/mg, about 95 U/mg, or about 100 U/mg.Exemplary conditions for performing in vitro sulfate release activityassay using heparin disaccharide as substrate are provided below.Typically, this assay measures the ability of I2S to release sulfateions from a naturally derived substrate, heparin disaccharide. Thereleased sulfate may be quantified by ion chromatography. In some cases,ion chromatography is equipped with a conductivity detector. As anon-limiting example, samples are first buffer exchanged to 10 mM Naacetate, pH 6 to remove inhibition by phosphate ions in the formulationbuffer. Samples are then diluted to 0.075 mg/ml with reaction buffer (10mM Na acetate, pH 4.4) and incubated for 2 hrs at 37° C. with heparindisaccharide at an enzyme to substrate ratio of 0.3 g I2S/100 gsubstrate in a 30 L reaction volume. The reaction is then stopped byheating the samples at 100° C. for 3 min. The analysis is carried outusing a Dionex IonPac AS 18 analytical column with an IonPac AG 18 guardcolumn. An isocratic method is used with 30 mM potassium hydroxide at1.0 mL/min for 15 minutes. The amount of sulfate released by the I2Ssample is calculated from the linear regression analysis of sulfatestandards in the range of 1.7 to 16.0 nmoles. The reportable value isexpressed as Units per mg protein, where 1 unit is defined as 1 moles ofsulfate released per hour and the protein concentration is determined byA280 measurements.

In some embodiments, the enzymatic activity of recombinant I2S proteinmay also be determined using various other methods known in the art suchas, for example, 4-MUF assay which measures hydrolysis of4-methylumbelliferyl-sulfate to sulfate and naturally fluorescent4-methylumbelliferone (4-MUF). In some embodiments, a desired enzymaticactivity, as measured by in vitro 4-MUF assay, of the producedrecombinant I2S protein is at least about 0.5 U/mg, 1.0 U/mg, 1.5 U/mg,2 U/mg, 2.5 U/mg, 3 U/mg, 4 U/mg, 5 U/mg, 6 U/mg, 7 U/mg, 8 U/mg, 9U/mg, 10 U/mg, 12 U/mg, 14 U/mg, 16 U/mg, 18 U/mg, or 20 U/mg. In someembodiments, a desired enzymatic activity, as measured by in vitro 4-MUFassay, of the produced recombinant I2S protein ranges from about 0-50U/mg (e.g., about 0-40 U/mg, about 0-30 U/mg, about 0-20 U/mg, about0-10 U/mg, about 2-50 U/mg, about 2-40 U/mg, about 2-30 U/mg, about 2-20U/mg, about 2-10 U/mg, about 4-50 U/mg, about 4-40 U/mg, about 4-30U/mg, about 4-20 U/mg, about 4-10 U/mg, about 6-50 U/mg, about 6-40U/mg, about 6-30 U/mg, about 6-20 U/mg, about 6-10 U/mg). Exemplaryconditions for performing in vitro 4-MUF assay are provided below.Typically, a 4-MUF assay measures the ability of an I2S protein tohydrolyze 4-methylumbelliferyl-sulfate (4-MUF-SO₄) to sulfate andnaturally fluorescent 4-methylumbelliferone (4-MUF). One milliunit ofactivity is defined as the quantity of enzyme required to convert onenanomole of 4-MUF-SO₄ to 4-MUF in one minute at 37° C. Typically, themean fluorescence units (MFU) generated by I2S test samples with knownactivity can be used to generate a standard curve, which can be used tocalculate the enzymatic activity of a sample of interest. Specificactivity may then calculated by dividing the enzyme activity by theprotein concentration.

In either example, the protein concentration of a recombinant I2Scomposition may be determined by any suitable method known in the artfor determining protein concentrations. In some cases, the proteinconcentration is determined by an ultraviolet light absorbance assay.Such absorbance assays are typically conducted at about a 280 nmwavelength (A₂₈₀).

Charge Profile

Purified recombinant I2S may be characterized by the charge profileassociated with the protein. Typically, protein charge profile reflectsthe pattern of residue side chain charges, typically present on thesurface of the protein. Charge profile may be determined by performingan ion exchange (IEX) chromatography (e.g., HPLC) assay on the protein.In some embodiments, a “charge profile” refers to a set of valuesrepresenting the amount of protein that elutes from an ion exchangecolumn at a point in time after addition to the column of a mobile phasecontaining an exchange ion.

Typically, a suitable ion exchange column is an anion exchange column.For example, a charge profile may be determined by strong anion exchange(SAX) chromatography using a high performance liquid chromatography(HPLC) system. In general, recombinant I2S adsorbs onto the fixedpositive charge of a strong anion exchange column and a gradient ofincreasing ionic strength using a mobile phase at a predetermined flowrate elutes recombinant I2S species from the column in proportion to thestrength of their ionic interaction with the positively charged column.More negatively charged (more acidic) I2S species elute later than lessnegatively charged (less acid) I2S species. The concentration ofproteins in the eluate are detected by ultraviolet light absorbance (at280 nm).

In some embodiments, recombinant I2S adsorbs at about pH 8.0 in 20 mMTRIS-HCl onto the fixed positive charge of a Mini Q PE column and agradient of increasing ionic strength using a mobile phase consisting of20 mM TRIS-HCL, 1 M sodium chloride, pH 8.0 at a flow rate of 0.8 ml/minelutes recombinant I2S species from the column in proportion to thestrength of their ionic interaction with the positively charged column.

In some embodiments, a charge profile may be depicted by a chromatogramof absorbance units versus time after elution from the HPLC column. Thechromatogram may comprise a set of one or more peaks, with each peak inthe set identifying a subpopulation of recombinant I2Ss of thecomposition that have similar surface charges.

In some embodiments, a purified I2S protein composition exhibits atleast six peaks in its charge profile. An exemplary charge profile ofI2S is depicted in the Examples section and in FIG. 11. As shown in FIG.11, six peaks are labeled (A to F) in the order of increasing negativecharge and decreasing contribution to total peak area of thechromatogram. In some embodiments, the charge profile for a purifiedrecombinant I2S composition contains a different number, size, shape ortime interval of peaks depending on the amount of negative or positivecharges on the surface of the protein. In some embodiments, arecombinant I2S composition has a charge profile that has fewer than 6(e.g., fewer than 5, 4, 3, or 2) peaks. In some embodiments, a chargeprofile of recombinant I2S may have 5, 4, 3, 2, or 1 peak(s). Forexample, any one, two, three, four, or five of peaks A, B, C, D, E, andF may be absent or reduced in a purified recombinant I2S proteincomposition. Typically, a charge profile is considered more homogenousif there are fewer peaks.

Glycan Mapping

In some embodiments, a purified recombinant I2S protein may becharacterized by their proteoglycan composition, typically referred toas glycan mapping. Without wishing to be bound by any theory, it isthought that glycan linkage along with the shape and complexity of thebranch structure may impact in vivo clearance, lysosomal targeting,bioavailability, and/or efficacy.

Typically, a glycan map may be determined by enzymatic digestion andsubsequent chromatographic analysis. Various enzymes may be used forenzymatic digestion including, but not limited to, suitableglycosylases, peptidases (e.g., Endopeptidases, Exopeptidases),proteases, and phosphatases. In some embodiments, a suitable enzyme isalkaline phosphatase. In some embodiments, a suitable enzyme isneuraminidase. Glycans (e.g., phosphoglycans) may be detected bychromatographic analysis. For example, phosphoglycans may be detected byHigh Performance Anion Exchange Chromatography with Pulsed AmperometricDetection (HPAE-PAD) or size exclusion High Performance LiquidChromatography (HPLC). The quantity of glycan (e.g., phosphoglycan)represented by each peak on a glycan map may be calculated using astandard curve of glycan (e.g., phosphoglycan), according to methodsknown in the art and disclosed herein.

In some embodiments, a purified I2S according to the present inventionexhibits a glycan map comprising seven peak groups indicative of neutral(peak group 1), mono-sialylated (peak group 2), di-sialylated (peakgroup 3), monophosphorylated (peak group 4), tri-sialylated (peak group5), tetra-sialylated (peak group 6), and diphosphorylated (peak group 7)I2S protein, respectively. Exemplary glycan maps of I2S are depicted inFIG. 10. In some embodiments, a purified recombinant I2S has a glycanmap that has fewer than 7 peak groups (e.g., a glycan map with 6, 5, 4,3, or 2 peaks groups). In some embodiments, a purified recombinant I2Shas a glycan map that has more than 7 peak groups (e.g., 8, 9, 10, 11,12 or more).

The relative amount of glycan corresponding to each peak group may bedetermined based on the peak group area relative to the correspondingpeak group area in a predetermined reference standard. In someembodiments, peak group 1 (neutral) may have the peak group area rangingfrom about 40-120% (e.g., about 40-115%, about 40-110%, about 40-100%,about 45-120%, about 45-115%, about 45-110%, about 45-105%, about45-100%, about 50-120%, about 50-110%) relative to the correspondingpeak group area in a reference standard. In some embodiments, peak group2 (monosialylated) may have the peak group area ranging from about80-140% (e.g., about 80-135%, about 80-130%, about 80-125%, about90-140%, about 90-135%, about 90-130%, about 90-120%, about 100-140%)relative to the corresponding peak group area in the reference standard.In some embodiments, peak group 3 (disialylated) may have the peak grouparea ranging from about 80-110% (e.g., about 80-105%, about 80-100%,about 85-105%, about 85-100%) relative to the corresponding peak grouparea in the reference standard. In some embodiments, peak group 4(monophosphorylated) may have the peak group area ranging from about100-550% (e.g., about 100-525%, about 100-500%, about 100-450%, about150-550%, about 150-500%, about 150-450%, about 200-550%, about200-500%, about 200-450%, about 250-550%, about 250-500%, about250-450%, or about 250-400%) relative to the corresponding peak grouparea in the reference standard. In some embodiments, peak group 5(tri-sialylated) may have the peak group area ranging from about 70-110%(e.g., about 70-105%, about 70-100%, about 70-95%, about 70-90%, about80-110%, about 80-105%, about 80-100%, or about 80-95%) relative to thecorresponding peak group area in the reference standard. In someembodiments, peak group 6 (tetra-sialylated) may have the peak grouparea ranging from about 90-130% (e.g., about 90-125%, about 90-120%,about 90-115%, about 90-110%, about 100-130%, about 100-125%, or about100-120%) relative to the corresponding peak group area in the referencestandard. In some embodiments, peak group 7 (diphosphorylated) may havewith the peak group area ranging from about 70-130% (e.g., about70-125%, about 70-120%, about 70-115%, about 70-110%, about 80-130%,about 80-125%, about 80-120%, about 80-115%, about 80-110%, about90-130%, about 90-125%, about 90-120%, about 90-115%, about 90-110%)relative to the corresponding peak group area in the reference standard.Various reference standards for glycan mapping are known in the art andcan be used to practice the present invention. Typically, peak group 7(diphosphorylated) corresponds to the level of di-M6P on the surface ofthe purified recombinant I2S protein.

It is contemplated that the glycosylation pattern of a purified I2Simpacts the lysosomal targeting. Various in vitro cellular uptake assaysare known in the art and can be used to practice the present invention.For example, to evaluate the uptake of I2S by M6P receptors, cellularuptake assays are performed using human fibroblasts expressing M6Preceptors on their surface. The internalized amount of I2S can bemeasured by a ELISA method. In some embodiments, a purified recombinantI2S protein according to the present invention is characterized withcellular uptake of greater than 70%, 75%, 80%, 85%, 90%, 95%, asdetermined by an in vitro uptake assay.

Peptide Mapping

In some embodiments, peptide mapping may be used to characterize aminoacid composition, post-translational modifications, and/or cellularprocessing; such as cleavage of a signal peptide, formylglycineconversion and/or glycosylation. Typically, a recombinant protein may bebroken into discrete peptide fragments, either through controlled orrandom breakage, to produce a pattern or peptide map. In some cases, apurified I2S protein may be first subjected to enzymatic digest prior toanalytic analysis. Digestion may be performed using a peptidase,glycoside hydrolase, phosphatase, lipase or protease and/or combinationsthereof, prior to analytic analysis. The structural composition ofpeptides may be determined using methods well known in the art.Exemplary methods include, but are not limited to, Mass spectrometry,Nuclear Magnetic Resonance (NMR) or HPLC.

Percent Formylglycine Conversion

Peptide mapping can be used to determine Percent FGly conversion. Asdiscussed above, I2S activation requires Cysteine (corresponding toposition 59 of the mature human I2S) to formylglycine conversion byformylglycine generating enzyme (FGE) as shown below:

Therefore, the percentage of formylglycine conversion (% FG) can becalculated using the following formula:

${\%\mspace{14mu}{FG}\mspace{14mu}\left( {{of}\mspace{14mu}{DS}} \right)} = {\frac{{Number}\mspace{14mu}{of}\mspace{14mu}{active}\mspace{14mu} I\; 2\; S\mspace{14mu}{molecules}}{{Number}\mspace{14mu}{of}\mspace{14mu}{total}\mspace{14mu}\left( {{active} + {inactive}} \right)\mspace{14mu} I\; 2\; S\mspace{14mu}{molecules}} \times 100}$

To calculate % FG, a recombinant I2S protein may be digested into shortpeptides using a protease (e.g., trypsin or chymotrypsin). Shortpeptides may be separated and characterized using, e.g., size exclusionHigh Performance Liquid Chromatography (HPLC). The peptide containingthe position corresponding to position 59 of the mature human I2S may becharacterized to determine if the Cys at position 59 was converted to aFGly as compared to a control (e.g., an I2S protein without FGlyconversion or an I2S protein with 100% FGly conversion). The amount ofpeptides containing FGly (corresponding to number of active I2Smolecules) and the total amount of peptides with both FGly and Cys(corresponding to number of total I2S molecules) may be determined basedon the corresponding peak areas and the ratio reflecting % FG can becalculated.

In some embodiments, a purified recombinant I2S protein according to thepresent invention has at least about 70% (e.g., at least about 77%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%) conversion of the cysteine residuecorresponding to Cys59 of human I2S (SEQ ID NO: 1) toC_(α)-formylglycine (FGly). In some embodiments, a purified recombinantI2S protein according to the present invention has substantially 100%conversion of the cysteine residue corresponding to Cys59 of human I2S(SEQ ID NO: 1) to C_(α)-formylglycine (FGly).

Sialic Acid Content

In some embodiments, a purified recombinant I2S protein may becharacterized by their sialic acid composition. Without wishing to bebound by theory, it is contemplated that sialic acid residues onproteins may prevent, reduce or inhibit their rapid in vivo clearancevia the asialoglycoprotein receptors that are present on hepatocytes.Thus, it is thought that recombinant proteins that have relatively highsialic acid content typically have a relatively long circulation time invivo.

In some embodiments, the sialic acid content of a purified recombinantI2S protein may be determined using methods well known in the art. Forexample, the sialic acid content of a recombinant I2S protein may bedetermined by enzymatic digestion and subsequent chromatographicanalysis. Enzymatic digestion may be accomplished using any suitablesialidase. In some cases, the digestion is performed by a glycosidehydrolase enzyme, such as neuraminidase. Sialic acid may be detected bychromatographic analysis such as, for example, High Performance AnionExchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD).The quantity of sialic acid in a recombinant I2S composition may becalculated using a standard curve of sialic acid, according to methodsknown in the art and disclosed herein.

In some embodiments, the sialic acid content of a purified recombinantI2S protein may be greater than 16 mol/mol. The units “mol/mol” in thecontext of sialic acid content refers to moles of sialic acid residueper mole of enzyme. In some cases, the sialic acid content of arecombinant I2S protein is or greater than about 16.5 mol/mol, about 17mol/mol, about 18 mol/mol, about 19 mol/mol, about 20 mol/mol, about 21mol/mol, about 22 mol/mol or more. In some embodiments, the sialic acidcontent of a purified recombinant I2S protein may be in a range betweenabout 16-20 mol/mol, 16-21 mol/mol, about 16-22 mol/mol, 16-23 mol/mol,16-24 mol/mol, about 16-25 mol/mol, about 17-20 mol/mol, 17-21 mol/mol,about 17-22 mol/mol, 17-23 mol/mol, 17-24 mol/mol, or about 17-25mol/mol.

Pharmaceutical Composition and Administration

Purified recombinant I2S protein may be administered to a HunterSyndrome patient in accordance with known methods. For example, purifiedrecombinant I2S protein may be delivered intravenously, subcutaneously,intramuscularly, parenterally, transdermally, or transmucosally (e.g.,orally or nasally)).

In some embodiments, a recombinant I2S or a pharmaceutical compositioncontaining the same is administered to a subject by intravenousadministration.

In some embodiments, a recombinant I2S or a pharmaceutical compositioncontaining the same is administered to a subject by intrathecaladministration. As used herein, the term “intrathecal administration” or“intrathecal injection” refers to an injection into the spinal canal(intrathecal space surrounding the spinal cord). Various techniques maybe used including, without limitation, lateral cerebroventricularinjection through a burrhole or cisternal or lumbar puncture or thelike. In some embodiments, “intrathecal administration” or “intrathecaldelivery” according to the present invention refers to IT administrationor delivery via the lumbar area or region, i.e., lumbar ITadministration or delivery. As used herein, the term “lumbar region” or“lumbar area” refers to the area between the third and fourth lumbar(lower back) vertebrae and, more inclusively, the L2-S1 region of thespine.

In some embodiments, a recombinant I2S or a pharmaceutical compositioncontaining the same is administered to the subject by subcutaneous(i.e., beneath the skin) administration. For such purposes, theformulation may be injected using a syringe. However, other devices foradministration of the formulation are available such as injectiondevices (e.g., the Inject-Ease™ and Genject™ devices); injector pens(such as the GenPen™); needleless devices (e.g., MediJector™ andBioJector™); and subcutaneous patch delivery systems.

In some embodiments, intrathecal administration may be used inconjunction with other routes of administration (e.g., intravenous,subcutaneously, intramuscularly, parenterally, transdermally, ortransmucosally (e.g., orally or nasally)).

The present invention contemplates single as well as multipleadministrations of a therapeutically effective amount of a recombinantI2S or a pharmaceutical composition containing the same describedherein. A recombinant I2S or a pharmaceutical composition containing thesame can be administered at regular intervals, depending on the nature,severity and extent of the subject's condition (e.g., a lysosomalstorage disease). In some embodiments, a therapeutically effectiveamount of a recombinant I2S or a pharmaceutical composition containingthe same may be administered periodically at regular intervals (e.g.,once every year, once every six months, once every five months, onceevery three months, bimonthly (once every two months), monthly (onceevery month), biweekly (once every two weeks), weekly, daily orcontinuously).

A recombinant I2S or a pharmaceutical composition containing the samecan be formulated with a physiologically acceptable carrier or excipientto prepare a pharmaceutical composition. The carrier and therapeuticagent can be sterile. The formulation should suit the mode ofadministration.

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions (e.g., NaCl), saline, buffered saline,alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzylalcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,amylose or starch, sugars such as mannitol, sucrose, or others,dextrose, magnesium stearate, talc, silicic acid, viscous paraffin,perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinylpyrolidone, etc., as well as combinations thereof. The pharmaceuticalpreparations can, if desired, be mixed with auxiliary agents (e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, coloring, flavoringand/or aromatic substances and the like) which do not deleteriouslyreact with the active compounds or interference with their activity. Insome embodiments, a water-soluble carrier suitable for intravenousadministration is used.

The composition or medicament, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thecomposition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. The compositioncan also be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose,magnesium carbonate, etc.

The composition or medicament can be formulated in accordance with theroutine procedures as a pharmaceutical composition adapted foradministration to human beings. For example, in some embodiments, acomposition for intravenous administration typically is a solution insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilizing agent and a local anesthetic to ease pain atthe site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water, saline or dextrose/water. Where thecomposition is administered by injection, an ampule of sterile water forinjection or saline can be provided so that the ingredients may be mixedprior to administration.

As used herein, the term “therapeutically effective amount” is largelydetermined based on the total amount of the therapeutic agent containedin the pharmaceutical compositions of the present invention. Generally,a therapeutically effective amount is sufficient to achieve a meaningfulbenefit to the subject (e.g., treating, modulating, curing, preventingand/or ameliorating the underlying disease or condition). For example, atherapeutically effective amount may be an amount sufficient to achievea desired therapeutic and/or prophylactic effect, such as an amountsufficient to modulate lysosomal enzyme receptors or their activity tothereby treat such lysosomal storage disease or the symptoms thereof(e.g., a reduction in or elimination of the presence or incidence of“zebra bodies” or cellular vacuolization following the administration ofthe compositions of the present invention to a subject). Generally, theamount of a therapeutic agent (e.g., a recombinant lysosomal enzyme)administered to a subject in need thereof will depend upon thecharacteristics of the subject. Such characteristics include thecondition, disease severity, general health, age, sex and body weight ofthe subject. One of ordinary skill in the art will be readily able todetermine appropriate dosages depending on these and other relatedfactors. In addition, both objective and subjective assays mayoptionally be employed to identify optimal dosage ranges.

A therapeutically effective amount is commonly administered in a dosingregimen that may comprise multiple unit doses. For any particulartherapeutic protein, a therapeutically effective amount (and/or anappropriate unit dose within an effective dosing regimen) may vary, forexample, depending on route of administration, on combination with otherpharmaceutical agents. Also, the specific therapeutically effectiveamount (and/or unit dose) for any particular patient may depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific pharmaceutical agentemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and/or rate of excretion or metabolism of thespecific fusion protein employed; the duration of the treatment; andlike factors as is well known in the medical arts.

Additional exemplary pharmaceutical compositions and administrationmethods are described in PCT Publication WO2011/163649 entitled “Methodsand Compositions for CNS Delivery of Iduronate-2-Sulfatase;” andprovisional application Ser. No. 61/618,638 entitled “Subcutaneousadministration of iduronate 2 sulfatase” filed on Mar. 30, 2012, theentire disclosures of both of which are hereby incorporated byreference.

It is to be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the enzyme replacement therapy andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed invention.

EXAMPLES Example 1 Recombinant I2S AF Capture and Purification Process

This example demonstrates a simplified downstream purification processmay be used to capture and purify recombinant I2S produced in serum-freemedium. An exemplary purification scheme is depicted in FIG. 1.

A cell line stably expressing an iduronate-2-sulfatase enzyme (I2S) andformylglycine generating enzyme (FGE) was developed. Generation andcharacterization of exemplary cell lines are described in the U.S.Provisional Application entitled “Cells for Producing RecombinantIduronate-2-Sulfatase” filed on even date herewith, the entire contentsof which is hereby incorporated by reference. Briefly, a human cell linewas engineered to co-express human I2S protein with the amino acidsequence shown in SEQ ID NO:2 and human formylglycine generating enzyme(FGE) with the amino acid sequence shown in SEQ ID NO:6.

> Full-length Precursor iduronate 2-sulfatase SEQ ID NO: 2MPPPRTGRGLLWLGLVLSSVCVALGSETQANSTTDALNVLLIIVDDLRPSLGCYGDKLVRSPNIDQLASHSLLFQNAFAQQAVCAPSRVSFLTGRRPDTTRLYDFNSYWRVHAGNFSTIPQYFKENGYVTMSVGKVFHPGISSNHTDDSPYSWSFPPYHPSSEKYENTKTCRGPDGELHANLLCPVDVLDVPEGTLPDKQSTEQAIQLLEKMKTSASPFFLAVGYHKPHIPFRYPKEFQKLYPLENITLAPDPEVPDGLPPVAYNPWMDIRQREDVQALNISVPYGPIPVDFQRKIRQSYFASVSYLDTQVGRLLSALDDLQLANSTIIAFTSDHGWALGEHGEWAKYSNFDVATHVPLIFYVPGRTASLPEAGEKLFPYLDPFDSASQLMEPGRQSMDLVELVSLFPTLAGLAGLQVPPRCPVPSFHVELCREGKNLLKHFRFRDLEEDPYLPGNPRELIAYSQYPRPSDIPQWNSDKPSLKDIKIMGYSIRTIDYRYTVWVGFNPDEFLANFSDIHAGELYFVDSDPLQDHNMYNDSQ GGDLFQLLMPFull-length human FGE precursor: SEQ ID NO: 6MAAPALGLVCGRCPELGLVLLLLLLSLLCGAAGSQEAGTGAGAGSLAGSCGCGTPQRPGAHGSSAAAHRYSREANAPGPVPGERQLAHSKMVPIPAGVFTMGTDDPQIKQDGEAPARRVTIDAFYMDAYEVSNTEFEKFVNSTGYLTEAEKFGDSFVFEGMLSEQVKTNIQQAVAAAPWWLPVKGANWRHPEGPDSTILHRPDHPVLHVSWNDAVAYCTWAGKRLPTEAEWEYSCRGGLHNRLFPWGNKLQPKGQHYANIWQGEFPVTNTGEDGFQGTAPVDAFPPNGYGLYNIVGNAWEWTSDWWTVHHSVEETLNPKGPPSGKDRVKKGGSYMCHRSYCYRYRCAARSQNTPDSSASN LGFRCAADRLPTMD

After synthesis of the full length I2S enzyme, the 25 amino acid signalpeptide is removed and a soluble mature I2S enzyme is secreted from thecell.

A chemically defined media (serum free/animal-component free; AF) wasused in the bioreactor process.

Individual harvest material was reduced in volume and buffer exchangedthrough an ultrafiltration/diafiltration process. The material, termedunpurified bulk (UPB), was frozen at −50° C. per individual harvest. Thedownstream purification process began with the thaw and poolofunpurified bulk and included successive viral inactivation, anionexchange (Capto Q), mixed mode (ceramic hydroxyapatite), cation exchange(SP Sepharose) and hydrophobic interaction (Phenyl Sepharose)chromatography steps followed by viral filtration, and finalconcentration and diafiltration step. In particular, this purificationprocess utilized Q, hydroxyapatite, SP and Phenyl chromatographicmodalities. Protein G Chromatography and Size Exclusion Chromatographytraditionally used in I2S purification process were removed. Exemplarysteps are shown in Table 3, as shown in FIG. 12.

Purified I2S protein were assessed for purity by peptide mapping,SDS-PAGE (Silver), size exclusion HPLC. Enzyme specific activity,formylglycine content, sialic acid content, glycan map, charge profileswere determined using standard methods. Exemplary results are shown inTable 4.

TABLE 4 Analysis of Purified Recombinant I2S Protein Purified I2S (10 Lscale) Assay Min-Max (n) Peptide Mapping L1 100-105% (n = 3) L10 98-100%(n = 3) L12 102-102% (n = 3) L13 96-97% (n = 3) L14 102-103% (n = 3) L17101-101% (n = 3) L20 102-103% (n = 3) Host Cell Protein ≦62.5 (n = 5)SDS-PAGE Conforms (Silver) Ion Exchange % Peak A 69-69% (n = 2) HPLC %Peak B 20-21% (n = 2) % Peak E + F 10-11% (n = 2) Size Exclusion99.9-99.9% (n = 5) HPLC Cellular Uptake 85, 95% and 97% (Bioassay) (n =3) % Formylglycine 87-95% (n = 5) Specific Activity 62-78 (n = 5) GlycanMap Pk Grp 3 88-93% (n = 5) Pk Grp 5 72-110% (n = 5) Pk Grp 6 124-133%(n = 5) Pk Grp 7 78-87% (n = 5) Total Area 94-116% (n = 5) Sialic Acid16-22 (n = 4) Endotoxin <0.04-<0.05 (n = 2) Bioburden 0.00-0.00 (n = 2)

An exemplary peptide map as compared to commercially available I2Sreference is shown in FIG. 2. Exemplary SDS-PAGE (Silver) analysisresults are shown in FIG. 3. Typically, using a process describedherein, the HCP concentration of drug substance (DS) was <100 ppm,meeting the <100 ppm specification required in many markets includingthe US. The SEC of DS was >99.5%, also meeting the current >99.3%marketing specification requirement in many markets. Exemplary chargeprofile is shown in FIG. 4. Exemplary glycan map is shown in FIG. 5. Inparticular, the glycan map of purified I2S includes seven peak groups,eluting according to an increasing amount of negative charges derivedfrom sialic acid and mannose-6-phosphate residues, representing in theorder of elution, neutrals, mono-, disialylated, monophosphorylated,trisialylated and hybrid (monosialylated and capped M6P),tetrasialylated and hybrid (disialylated and capped M6P) anddiphosphorylated glycans.

Taken together, this example demonstrates that a simplified four-columnpurification process can be used to successfully purify recombinant I2Sproduced in animal free medium at large scale.

Example 2 Harvest and Viral Inactivation Stability Studies ofRecombinant I2S AF

The objective of this study was to evaluate the effects of temperaturehold time and freeze-thaw cycles on the stability of recombinant I2Sclarified harvest.

Clarified harvest samples were stored at ambient and 2-8° C. for up toseven days and the viral inactivated UPB samples were held at ambientfor up to 24 hours. Freeze-thaw samples on clarified harvests werefrozen at −20° C., −50° C., and −80° C. and experienced freeze-thaw forup to three cycles. Stability was gauged using Western blot, SEC HPLC,and activity assay.

I2S-AF harvest material was produced from the 2D cell line by CCPD usinga B. Braun 20 L bioreactor with a centrifuge retention device and adesired bleeding rate. For the temperature holding study, each clarifiedharvest was stored at ambient and 2-8° C. and sampled at selected holdtimes. Sampling amounts and hold times are listed in Table 5.Freeze-thaw samples were stored at −20° C., −50° C., and −80° C. andthawed using a water bath at 25° C.

TABLE 5 Clarified Harvest Hold Point Stability Holding Time SamplesHolding Temperature (Days) Clarified 15 × 0.5 mL 2-8° C. T = 0, 24 h, 76h, Harvest 12 120 h, 168 h 15 × 0.5 mL Ambient T = 0, 24 h, 76 h, 120 h,168 h  9 × 0.5 mL −20° C., −50° C., Freeze/Thaw 1, 2, and −80° C. and 3Clarified 15 × 0.5 mL 2-8° C. T = 0, 24 h, 76 h, Harvest 18 120 h, 168 h15 × 0.5 mL Ambient T = 0, 24 h, 76 h, 120 h, 168 h  9 × 0.5 mL −20° C.,−50° C., Freeze/Thaw 1, 2, and −80° C. and 3

The viral inactivation step occurred at the unpurified bulk step priorto loading the first column. UPB was produced by concentrating andbuffer exchange of clarified harvest. UF/DF was performed using a Pall 1sq. ft. Centramate system and buffer exchanged into 10 mM MES, 155 mMNaCl, pH=6.5. The viral inactivation step added 1% Tween 80 and 0.3%TnBP, filtered using Durapore syringe filters for each time point.Samples were taken at each time point listed in Table 6 and frozen at−80° C. Samples from the clarified harvest hold point and freeze-thawstudies were tested by Western blot and activity (4-MU assay). UPBsamples from the viral inactivation were tested for purity by SEC HPLC.The hold point activity results from Harvest 12 and 18 on Table 5 showedno significant changes up to 7 days of storage at ambient and 2-8° C.for both harvests. There were no significant changes seen in Harvest 12activity for up to 3 freeze-thaw cycles stored at −20° C., −50° C., and−80° C.

TABLE 6 Viral Inactivation of Unpurified Bulk Holding SamplesTemperature Holding Time (Days) Viral 9 × 0.5 mL Ambient, Control T = 0,6 h, 24 h Inactivation 9 × 0.5 mL Ambient, Viral T = 0, 6 h, 24 hInactivation

Activity and SEC-HPLC for the stability of the viral inactivation UPBstep are described in FIGS. 6 and 7. This shows that there were noissues in viral inactivation stability based on activity and purity forup to 24 hours.

In summary, based on the stability analysis described herein, clarifiedharvest can be stored at 2-8° C. (for example, for up to 7 days) withoutsignificant changes in harvest quality. Clarified harvests canexperience multiple freeze-thaw cycles and stored at −20° C., −50° C.,and −80° C. temperatures with no significant changes in stability. Basedon SEC HPLC purity results, viral inactivation at the UPB step can occurat ambient temperature (e.g., for up to 24 hours) with no changes inactivity and purity.

Example 3 Purification and Analysis of Animal-Free IL CD MediaConfirmation Run

The objective of this study was to perform purification from pooledharvest of I2S-AF produced in an animal-free perfusion using chemicallydefined media and to characterize the drug substance.

This study evaluated I2S-AF purification process performance and drugsubstance (DS) produced from a chemically defined medium bioreactor.

Cell Culture

The I2S-AF material was produced from cell line 2D expressing I2S andformylglycine generating enzyme (FGE)) as described in Example 1. Thematerial was produced in CCPD in a 1 L Das Gip spin filter bioreactorusing a chemically defined serum free media. Individual bags from eachclarified harvest (HI-21) were received frozen at −20° C. and thawed at2-8° C. overnight. Equal volumes of each clarified harvest was pooled torepresent an entire harvest pool, then 0.2 μm filtered and concentratedusing 30 kD Pall Omega Centramate cassette with a total membrane area of1 ft². The unpurified bulk (UPB) was 0.2 um filtered and frozen prior touse.

Purification

Exemplary column specifications and loading are described in Table 7.The Q Sepharose was loaded at a target of 3 g/L by titer. Subsequentcolumns were loaded at 100% from the previous column elution and nomaterial removed.

TABLE 7 Column and Loading Specifications Column Column Column LoadDimensions Volume (g/L resin by Column Column (cm × cm) (mL) I2S) Load(mg) Q Sepharose 2.6 × 25 133 3 399 HA Type II, 1.6 × 30 60 5.5 330 80μm Phenyl Sepharose 1.6 × 23 46 5.6 258

One purification run was performed using UPB from pooling harvests 1through 21 from the bioreactor. UPB was thawed at 2-8° C. overnight andpooled by equal volume from each harvest.

Individual column process steps and buffer formulations can be found inTables 8-11. The pooled UPB was filtered using a 0.2 um bottle filtersystem, adjusted to pH 6.5 using 1 M sodium acetate, and conductivityadjusted to 16 mS/cm with 5 M sodium chloride prior to loading onto theQ Sepharose FF column. The Q Sepharose elution was adjusted to 0.001 MNaP0₄ using 0.25M NaPO₄, pH 5.5 and filtered with a 0.22 um PES bottletop filter prior to loading onto the HA column. The HA elutionconductivity was adjusted to 1.55 M NaCl with 5 M NaCl and pH adjustedto pH 5.5 with 1 M sodium acetate. The adjustment time was approximately1 hour. The adjusted pool was filtered using a 0.22 um PES bottle topfilter prior to loading onto the Phenyl Sepharose column. The Phenylelution was concentrated 4× and diafiltered 6× into 0.02 M NaPO₄, 0.137M NaCl, pH 6.0. The diafiltered product was adjusted to 2.0 g/L andformulated with 0.2% Polysorbate 20 to generate mock drug substance. Amock pool of H1-20 of the DS was created for additionalcharacterization.

TABLE 8 Exemplary Process Details for Q Sepharose FF Chromatography Flowrate Process Step (cm/hr) CV Buffers Sanitization 150 3 0.5N NaOHEquilibration 150 4 0.01M MES, 0.155M NaCl, ph 6.5 Wash 1 150 2 0.01MMES, 0.155M NaCl, ph 6.5 Wash 2 150 3 0.01M MES, 0.155M NaCl, ph 5.5Elution 150 3 0.01M MES, 0.50M NaCl, ph 5.5 Clean/Strip 150 4 1.0M NaOH,2M NaCl Store 150 4 0.0N NaOH

TABLE 9 Exemplary Process Details for HA Chromatography Process StepFlow rate (cm/hr) CV Buffers Sanitization 200 3 0.5N NaOH Charge 200 30.250M NaPO₄, pH 5.5 Equilibration 200 3-6 0.01M MES, 0.001M NaPO₄, 0.5MNaCl, pH 5.5 Wash 1 200 1 0.01M MES, 0.001M NaPO₄, 0.5M NaCl, pH 5.5Wash 2 200 6 0.01M MES, 0.01M NaPO₄, 0.5M NaCl, pH 5.5 Elution 200 30.01M MES, 0.08M NaPO₄, pH 5.5 Strip 200 4 0.4M NaPO₄ pH 12 Clean 200 40.5N NaOH Store 200 4 0.1N NaOH

TABLE 10 Exemplary Process Details for Phenyl Sepharose ChromatographyProcess Step Flow rate (cm/hr) CV Buffers Sanitization 150 3 0.5N NaOHEquilibration 150 4-6 0.02M MES, 1.5M NaCl, pH 5.5 Wash 150 2 0.02M MES,1.5M NaCl, pH 5.5 Elution 150 3 0.02M MES, 0.2M NaCl, pH 5.5 Water Wash150 3 RO/DI Water Ethanol 150 3 20% Ethanol Wash Clean 150 3 0.5N NaOHStore 150 3 0.01N NaOH

TABLE 11 Exemplary Diafiltration of the Phenyl Elution Pool FiltrationUnit Centricon Plus 70 Diafiltration Buffer 0.02M NaPO₄, 0.137M NaCl, pH6.0 Diafiltration Volumes 6X-8XIn Process Purity by HCP by ELISA

Table 12 describes the in-process HCP removal for each step. Thein-process HCP results were high with the majority of removal at the HAstep.

TABLE 12 In-Process HCP Removal Step HCP (ng/mg) LRV HCP Fold Q 46,3920.3 2 51,957 HA 51,957 1.3 18 5,876 Phenyl 5,876 0.7 5 1,870Drug Substance Characterization

Exemplary drug substance lot release results are listed in Table 13. Ascan be seen, the drug substance had high specific activity and % FG inthe purified material. Exemplary drug attributes characterization isshown in Table 13. HCP was reduced from 1,870 ng/mg to 372 ng/mg at thefinal UF/DF step.

TABLE 13 Exemplary Drug Substance Lot Release 1 L CD media DS LotRelease (I2S-AF) % FG 94% Glycan Map Group 3 99% Group 5 89% Group 6104%  Group 7 (2-M6P) 95% Total Area 107%  Sialic Acid 17Internalization 83% SEC-HPLC 99.9%   Specific Activity 82 (U/mg) IEXHPLC A (%) 64% B (%) 23% A + B 87% E + F  0% Host Cell Protein 372  CellUptake 98

Example 4 Physiochemical and Biological Characterization of PurifiedRecombinant I2S Enzyme

The purpose of the example was to perform a detailed characterization ofthe recombinant I2S protein purified using methods described above.

SDS-PAGE

For the experiment, recombinant I2S protein was generated using the 2Dand 4D human cell lines, in two separate serum-free cell culturereactions. Samples were collected and purified using methods describedabove. Purified I2S enzyme was analyzed by SDS-PAGE, and treated withsilver stain for visualization. Exemplary results are shown in FIG. 8.As can be seen from FIG. 8, purified recombinant I2S protein usingmethods described herein present comparable banding patterns as comparedto the I2S reference sample purified using standard method.

Peptide Map

Recombinant I2S protein produced by the I2S-AF 2D cell line was purifiedusing methods as described above. Purified recombinant I2S and a sampleof reference human I2S were each subjected to proteolytic digest andexamined by HPLC analysis. An exemplary peptide map as compared to thatof a reference I2S is shown in FIG. 9.

Percent Formylglycine Conversion

Peptide mapping can be used to determine Percent FGly conversion. I2Sactivation requires Cysteine (corresponding to position 59 of the maturehuman I2S) to formylglycine conversion by formylglycine generatingenzyme (FGE) as shown below:

Therefore, the percentage of formylglycine conversion (% FG) can becalculated using the following formula:

${\%\mspace{14mu}{FG}\mspace{14mu}\left( {{of}\mspace{14mu}{DS}} \right)} = {\frac{{Number}\mspace{14mu}{of}\mspace{14mu}{active}\mspace{14mu} I\; 2\; S\mspace{14mu}{molecules}}{{Number}\mspace{14mu}{of}\mspace{14mu}{total}\mspace{14mu}\left( {{active} + {inactive}} \right)\mspace{14mu} I\; 2\; S\mspace{14mu}{molecules}} \times 100}$

For example 50% FG means half of the purified recombinant I2S isenzymatically inactive without any therapeutic effect.

Peptide mapping was used to calculate % FG. Briefly, a purifiedrecombinant I2S protein was digested into short peptides using aprotease (e.g., trypsin or chymotrypsin). Short peptides were separatedand characterized using HPLC. The peptide containing the positioncorresponding to position 59 of the mature human I2S was characterizedto determine if the Cys at position 59 was converted to a FGly ascompared to a control (e.g., an I2S protein without FGly conversion oran I2S protein with 100% FGly conversion). The amount of peptidescontaining FGly (corresponding to number of active I2S molecules) andthe total amount of peptides with both FGly and Cys (corresponding tonumber of total I2S molecules) may be determined based on thecorresponding peak areas and the ratio reflecting % FG was calculated.Exemplary results are shown in Table 14.

Glycan Map—Mannose-6-Phosphate and Sialic Acid Content

The glycan and sialic acid composition of purified recombinant I2Sprotein was determined. Quantification of the glycan composition wasperformed, using anion exchange chromatography to produce a glycan map.As described below, the glycan map of recombinant I2S purified underconditions described herein consists of seven peak groups, elutingaccording to an increasing amount of negative charges, at least partlyderived from sialic acid and mannose-6-phosphate glycoforms resultingfrom enzymatic digest. Briefly, purified recombinant I2S from theserum-free cell culture (I2S-AF 2D Serum-free and I2S-AF 4D Serum-free)and reference recombinant I2S, were treated with either (1) purifiedneuraminidase enzyme (isolated from Arthrobacter Ureafaciens (10 mU/μL),Roche Biochemical (Indianapolis, Ind.), Cat. #269 611 (1 U/100 μL)) forthe removal of sialic acid residues, (2) alkaline phosphatase for 2hours at 371° ° C. for complete release of mannose-6-phosphate residues,(3) alkaline phosphatase+neuraminidase, or (4) no treatment. Eachenzymatic digest was analyzed by High Performance Anion ExchangeChromatography with Pulsed Amperometric Detection (HPAE-PAD) using aCarboPac PA1 Analytical Column equipped with a Dionex CarboPac PA1 GuardColumn. A series of sialic acid and mannose-6-phosphate standards in therange of 0.4 to 2.0 nmoles were run for each assay. An isocratic methodusing 48 mM sodium acetate in 100 mM sodium hydroxide was run for aminimum of 15 minutes at a flow rate of 1.0 mL/min at ambient columntemperature to elute each peak. The data generated from each individualrun, for both the I2S-AF and reference I2S samples, were each combinedinto a single chromatograph to represent the glycan map for eachrespective recombinant protein. As indicated in FIG. 10, the glycan mapfor I2S purified from serum-free medium showed representative elutionpeaks (in the order of elution) constituting neutrals, monosialylated,disialylated, monophosphorylated, trisialylated and hybrid(monosialylated and capped mannose-6-phosphate), tetrasialylated andhybrid (disialylated and capped mannose-6-phosphate) anddiphosphorylated glycans. Exemplary glycan maps are shown in FIG. 10.

Average sialic acid content (moles sialic acid per mole protein) in eachrecombinant I2S sample was calculated from linear regression analysis ofsialic acid standards. Each chromatogram run was visualized using thePeakNet 6 Software. Sialic acid standards and sialic acid released fromrecombinant I2S assay control and test samples appear as a single peak.The amount of sialic acid (nmoles) for I2S was calculated as a raw valueusing the following equation:

${S.A.\mspace{14mu}\left( {{mole}\mspace{14mu}{per}\mspace{14mu}{mole}\mspace{14mu} I\; 2S} \right)} = \frac{\left( {{nmoles}\mspace{14mu}{sialic}\mspace{14mu}{acid}} \right)}{(0.3272)(C)}$Where C is the protein concentration (in mg/ml) of sample or recombinantI2S assay control.The corrected value of sialic acid as moles of sialic acid per mole ofprotein for each test sample was calculated using the following formula:

${{Corrected}\mspace{14mu}{S.A.}} = \frac{\begin{matrix}{\left( {{Sample}\mspace{14mu}{Raw}\mspace{14mu}{Sialic}\mspace{14mu}{Acid}\mspace{14mu}{Value}} \right) \times} \\\left( {{Established}\mspace{14mu}{Idursulfase}\mspace{14mu}{Assay}\mspace{14mu}{Control}\mspace{14mu}{Value}} \right)\end{matrix}}{\left( {{Idursulfase}\mspace{14mu}{Assay}\mspace{14mu}{Control}\mspace{20mu}{Raw}\mspace{14mu}{Sialic}\mspace{14mu}{Acid}\mspace{11mu}{Value}} \right)}$

Exemplary data indicative of sialic acid content on the recombinant I2Spurified from I2S-AF 2D or 4D cell lines are shown in Table 14.

TABLE 14 Exemplary Characteristics of I2S Purified from Serum-Free CellCulture I2S-AF 2D Assay (Serum-free) Peptide Mapping L1 101 L10 100 L12102 L13 97 L14 101 L17 100 L20 102 Host Cell Protein <62.5 ng/mg IonExchange HPLC % Area Peak A 62 Peak A + B 82 Peak E + F 0 %Formylglycine 87 Specific activity (U/mg) 64 (sulfate release assay) %Size Exclusion ≧99.8 (n = 13) HPLC Glycan Mapping Monosialylated 105Disialylated 93 Monophosphorylated 139 Trisialylated 89 Tetrasialylated125 Diphosphorylated 95 Sialic Acid (mol/mol) 20Specific Activity

Specific activity of the recombinant I2S enzyme purified using methodsdescribed herein was analyzed using in vitro sulfate release assay or4-MUF assay.

In Vitro Sulfate Release Assay

In vitro sulfate release activity assay was conducted using heparindisaccharide as substrate. In particular, this assay measures theability of I2S to release sulfate ions from a naturally derivedsubstrate, heparin disaccharide. The released sulfate may be quantifiedby ion chromatography equipped with a conductivity detector. Briefly,samples were first buffer exchanged to 10 mM Na acetate, pH 6 to removeinhibition by phosphate ions in the formulation buffer. Samples werethen diluted to 0.075 mg/ml with reaction buffer (10 mM Na acetate, pH4.4) and incubated for 2 hrs at 37° C. with heparin disaccharide at anenzyme to substrate ratio of 0.3 μg I2S/100 μg substrate in a 30 Lreaction volume. The reaction was then stopped by heating the samples at100° C. for 3 min. The analysis was carried out using a Dionex IonPac AS18 analytical column with an IonPac AG 18 guard column. An isocraticmethod was used with 30 mM potassium hydroxide at 1.0 mL/min for 15minutes. The amount of sulfate released by the I2S sample was calculatedfrom the linear regression analysis of sulfate standards in the range of1.7 to 16.0 nmoles. The reportable value was expressed as Units per mgprotein, where 1 unit is defined as 1 μmoles of sulfate released perhour and the protein concentration is determined by A280 measurements.Exemplary results are shown in Table 14.

4-MUF Assay

Specific activity of the purified recombinant I2S enzyme may also beanalyzed using the fluorescence based 4-MUF assay. Briefly, the assaymeasures the hydrolysis of I2S substrate 4-methylumbelliferyl-sulfate(4-MUF-SO₄). Upon cleavage of the 4-MUF-SO₄ substrate by I2S, themolecule is converted to sulfate and naturally fluorescent4-methylumbelliferone (4-MUF). As a result, I2S enzyme activity can bedetermined by evaluating the overall change in fluorescent signal overtime. For this experiment, purified I2S enzyme were incubated with asolution of 4-methylumbelliferyl-sulfate (4-MUF-SO₄), Potassium Salt,Sigma Cat. #M-7133). Calibration of the assay was performed using aseries of control reference samples, using commercially available I2Senzyme diluted at 1:100, 1:200 and 1:20,000 of the stock solution. Theenzymatic assay was run at 37° C. and assayed using a calibratedfluorometer. Using the fluorescence values obtained for each referencestandard, the percent coefficient of variation was determined using thefollowing equation:

${\%\mspace{14mu}{CV}} = {\frac{{Standard}\mspace{14mu}{Deviation}\mspace{14mu}{of}\mspace{20mu}{Raw}\mspace{14mu}{Fluorescenc}\underset{\_}{e}\mspace{14mu}{Values}\mspace{14mu}\left( {N = 3} \right)}{{Average}\mspace{14mu}{Fluorescence}\mspace{14mu}{Value}} \times 100\%}$

The percent CV values were then used to calculate the Corrected AverageFluorescence for each sample, in order to determine the reportableenzyme activity, expressed in mU/mL using the following formula:

${{mU}\text{/}{mL}} = {\left( {C\; F\; U} \right)\left( \frac{1\mspace{14mu}{nmole}\text{/}L}{10\mspace{14mu}{FU}} \right)\left( \frac{1\mspace{14mu} L}{10^{3}\mspace{14mu}{mL}} \right)\left( \frac{2.11\mspace{14mu}{mL}}{0.01\mspace{14mu}{mL}} \right)\left( \frac{1\mspace{14mu}{hour}}{60\mspace{14mu}\min} \right)\left( \frac{1\mspace{14mu}{mU}}{nmole} \right)({DF})}$C F U = Negative  corrected  average  fluorescenceD F − Dilution  Factor

One milliunit of activity is the quantity of enzyme required to convert1 nanomole of 4-methylumbelliferyl-sulfate to 4-methylumbelliferone in 1minute at 37° C.

Charge Profile

For this experiment, the charge distribution of each purifiedrecombinant I2S was determined by Strong Anion Exchange (SAX)Chromatography, with a High Performance Liquid Chromatography (HPLC)system. The method separates recombinant I2S variants within the sample,based on surface charge differences. At pH 8.00, negatively chargedspecies adsorb onto the fixed positive charge of the SAX column. Agradient of increasing ionic strength is used to elute each proteinspecies in proportion to the strength of their ionic interaction withthe column. One hundred micrograms of purified I2S, isolated from the 2Dcell line under serum-free growth conditions or reference recombinantI2S enzyme, was loaded onto an Amersham Biosciences Mini Q PE (4.6×50mm) column held at ambient temperature and equilibrated to 20 mMTris-HCl, pH 8.00. Gradient elution was made at a flow rate of 0.80mL/min, using a mobile phase of 20 mM Tris-HCl, 1.0 M sodium chloride,pH 8.00. Protein concentration was continuously determined during therun, by measuring light absorbance of the sample elution at the 280 nmwavelength. Exemplary results showing charge profiles observed forrecombinant I2S purified from 2D and 4D cell lines are shown in FIG. 11.

What is claimed is:
 1. A method of treating Hunter syndrome comprisingadministering to a patient a pharmaceutical composition comprisingpurified recombinant iduronate-2-sulfatase (I2S) having an amino acidsequence with at least 90% sequence identity to SEQ ID NO: 1 and acarrier, wherein the purified recombinant I2S comprises at least 70%conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1to Ca-formylglycine (FGly), and wherein the purified recombinant I2Scontains less than 150 ng Host Cell Protein (HCP)/mg I2S.
 2. The methodof claim 1, wherein the purified recombinant I2S comprises at least 75%conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1to Ca-formylglycine (FGly).
 3. The method of claim 1, wherein thepurified recombinant I2S comprises at least 85% conversion of thecysteine residue corresponding to Cys59 of SEQ ID NO:1 toCa-formylglycine (FGly).
 4. The method of claim 1, wherein thepharmaceutical composition is administered by intrathecal or intravenousinjection.
 5. The method of claim 4, wherein the pharmaceuticalcomposition is administered by intravenous infusion.
 6. The method ofclaim 1, wherein the pharmaceutical composition is administered onceweekly.
 7. The method of claim 1, wherein the pharmaceutical compositionis administered biweekly.
 8. The method of claim 1, wherein thepharmaceutical composition is administered once monthly.
 9. The methodof claim 1, wherein administration of the pharmaceutical compositionresults in a reduction of zebra bodies in the patient.
 10. The method ofclaim 1, wherein administration of the pharmaceutical compositionresults in a reduction in the amount of glucosaminoglycans withinlysosomes of the patient.
 11. A method of treating Hunter syndromecomprising administering to a patient a pharmaceutical compositioncomprising purified recombinant iduronate-2-sulfatase (I2S) having anamino acid sequence with at least 90% sequence identity to SEQ ID NO: 1and a carrier, wherein the purified recombinant I2S comprises at least70% conversion of the cysteine residue corresponding to Cys59 of SEQ IDNO:1 to Ca-formylglycine (FGly), and wherein the purified recombinantI2S contains at least 10% bis-phosphorylated oligosaccharides permolecule.
 12. The method of claim 11, wherein the purified recombinantI2S contains at least 20% bis-phosphorylated oligosaccharides permolecule.
 13. The method of claim 11, wherein the pharmaceuticalcomposition is administered by intrathecal or intravenous injection. 14.The method of claim 13, wherein the pharmaceutical composition isadministered by intravenous infusion.
 15. The method of claim 11,wherein administration of the pharmaceutical composition results in areduction of zebra bodies in the patient.
 16. The method of claim 11,wherein administration of the pharmaceutical composition results in areduction in the amount of glucosaminoglycans within lysosomes of thepatient.
 17. A method of treating Hunter syndrome comprisingadministering to a patient a pharmaceutical composition comprisingpurified recombinant iduronate-2-sulfatase (I2S) having an amino acidsequence with at least 90% sequence identity to SEQ ID NO: 1 and acarrier, wherein the purified recombinant I2S comprises at least 70%conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1to Ca-formylglycine (FGly), and wherein the purified recombinant I2Sprotein has specific activity of at least 40 U/mg as determined by an invitro sulfate release activity assay using heparin disaccharide assubstrate.
 18. A method of treating Hunter syndrome comprisingadministering to a patient a pharmaceutical composition comprisingpurified recombinant iduronate-2-sulfatase (I2S) having an amino acidsequence with at least 90% sequence identity to SEQ ID NO: 1 and acarrier, wherein the purified recombinant I2S comprises at least 70%conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1to Ca-formylglycine (FGly), and wherein the purified recombinant I2Sprotein has specific activity of at least 20 U/mg as determined by an invitro 4-MUF-S04 to 4-MUF conversion assay.
 19. A method of treatingHunter syndrome comprising administering to a patient a pharmaceuticalcomposition comprising purified recombinant iduronate-2-sulfatase (I2S)having an amino acid sequence with at least 90% sequence identity to SEQID NO: 1 and a carrier, wherein the purified recombinant I2S comprises:(i) at least about 70% conversion of the cysteine residue correspondingto Cys59 of SEQ ID NO:1 to Ca-formylglycine (FGly), and (ii) on averageat least 16 sialic acids per molecule.
 20. A method of treating Huntersyndrome comprising administering to a patient a pharmaceuticalcomposition comprising purified recombinant iduronate-2-sulfatase (I2S)having an amino acid sequence with at least 90% sequence identity to SEQID NO: 1 and a carrier, wherein the purified recombinant I2S comprisesat least 70% conversion of the cysteine residue corresponding to Cys59of SEQ ID NO:1 to Ca-formylglycine (FGly), and wherein the purified I2Sis characterized with a glycan map comprising seven or fewer peak groupsselected from the peak groups indicative of neutral (peak group 1),mono-sialylated (peak group 2), di-sialylated (peak group 3),monophosphorylated (peak group 4), tri-sialylated (peak group 5),tetra-sialylated (peak group 6), or diphosphorylated (peak group 7) I2Sprotein.